Rolling circle replication reporter systems

ABSTRACT

Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/038,718, filed on Jan. 2, 2002 now U.S. Pat. No. 6,797,474, which isa continuation of U.S. application Ser. No. 09/644,723, filed on Aug.23, 2000 now U.S. Pat. No. 6,344,329, which is a continuation of U.S.patent application Ser. No. 09/132,553, filed Aug. 11, 1998 now U.S.Pat. No. 6,210,844, which is a continuation of U.S. patent applicationSer. No. 08/563,912, filed Nov. 21, 1995 now U.S. Pat. No. 5,854,033,all of which are hereby incorporated by this reference in theirentirety.

BACKGROUND OF THE INVENTION

The disclosed invention is generally in the field of assays fordetection of analytes, and specifically in the field of nucleic acidamplification and detection.

A number of methods have been developed which permit the implementationof extremely sensitive diagnostic assays based on nucleic aciddetection. Most of these methods employ exponential amplification oftargets or probes. These include the polymerase chain reaction (PCR),ligase chain reaction (LCR), self-sustained sequence replication (3SR),nucleic acid sequence based amplification (NASBA), strand displacementamplification (SDA), and amplification with Qβ replicase (Birkenmeyerand Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren,Trends Genetics, 9:199-202 (1993)).

While all of these methods offer good sensitivity, with a practicallimit of detection of about 100 target molecules, all of them sufferfrom relatively low precision in quantitative measurements. This lack ofprecision manifests itself most dramatically when the diagnostic assayis implemented in multiplex format, that is, in a format designed forthe simultaneous detection of several different target sequences.

In practical diagnostic applications it is desirable to assay for manytargets simultaneously. Such multiplex assays are typically used todetect five or more targets. It is also desirable to obtain accuratequantitative data for the targets in these assays. For example, it hasbeen demonstrated that viremia can be correlated with disease status forviruses such as HIV-1 and hepatitis C (Lefrere et al., Br. J. Haematol.,82(2):467-471 (1992), Gunji et al., Int. J. Cancer, 52(5):726-730(1992), Hagiwara et al., Hepatology, 17(4):545-550 (1993), Lu et al., J.Infect. Dis., 168(5):1165-8116 (1993), Piatak et al., Science,259(5102):1749-1754 (1993), Gupta et al., Ninth International Conferenceon AIDS/Fourth STD World Congress, Jun. 7-11, 1993, Berlin, Germany,Saksela et al., Proc. Natl. Acad. Sci. USA, 91(3):1104-1108 (1994)). Amethod for accurately quantitating viral load would be useful.

In a multiplex assay, it is especially desirable that quantitativemeasurements of different targets accurately reflect the true ratio ofthe target sequences. However, the data obtained using multiplexed,exponential nucleic acid amplification methods is at bestsemi-quantitative. A number of factors are involved:

-   1. When a multiplex assay involves different priming events for    different target sequences, the relative efficiency of these events    may vary for different targets. This is due to the stability and    structural differences between the various primers used.-   2. If the rates of product strand renaturation differ for different    targets, the extent of competition with priming events will not be    the same for all targets.-   3. For reactions involving multiple ligation events, such as LCR,    there may be small but significant differences in the relative    efficiency of ligation events for each target sequence. Since the    ligation events are repeated many times, this effect is magnified.-   4. For reactions involving reverse transcription (3SR, NASBA) or    klenow strand displacement (SDA), the extent of polymerization    processivity may differ among different target sequences.-   5. For assays involving different replicatable RNA probes, the    replication efficiency of each probe is usually not the same, and    hence the probes compete unequally in replication reactions    catalyzed by Qβ replicase.-   6. A relatively small difference in yield in one cycle of    amplification results in a large difference in amplification yield    after several cycles. For example, in a PCR reaction with 25    amplification cycles and a 10% difference in yield per cycle, that    is, 2-fold versus 1.8-fold amplification per cycle, the yield would    be 2.0²⁵=33,554,000 versus 1.8²⁵=2,408,800. The difference in    overall yield after 25 cycles is 14-fold. After 30 cycles of    amplification, the yield difference would be more than 20-fold.

Accordingly, there is a need for amplification methods that are lesslikely to produce variable and possibly erroneous signal yields inmultiplex assays.

It is therefore an object of the disclosed invention to provide a methodof amplifying diagnostic nucleic acids with amplification yieldsproportional to the amount of a target sequence in a sample.

It is another object of the disclosed invention to provide a method ofdetecting specific target nucleic acid sequences present in a samplewhere detection efficiency is not dependent on the structure of thetarget sequences.

It is another object of the disclosed invention to provide a method ofdetermining the amount of specific target nucleic acid sequences presentin a sample where the signal level measured is proportional to theamount of a target sequence in a sample and where the ratio of signallevels measured for different target sequences substantially matches theratio of the amount of the different target sequences present in thesample.

It is another object of the disclosed invention to provide a method ofdetecting and determining the amount of multiple specific target nucleicacid sequences in a single sample where the ratio of signal levelsmeasured for different target nucleic acid sequences substantiallymatches the ratio of the amount of the different target nucleic acidsequences present in the sample.

It is another object of the disclosed invention to provide a method ofdetecting the presence of single copies of target nucleic acid sequencesin situ.

It is another object of the disclosed invention to provide a method ofdetecting the presence of target nucleic acid sequences representingindividual alleles of a target genetic element.

SUMMARY OF THE INVENTION

Disclosed are compositions and a method for amplifying nucleic acidsequences based on the presence of a specific target sequence oranalyte. The method is useful for detecting specific nucleic acids oranalytes in a sample with high specificity and sensitivity. The methodalso has an inherently low level of background signal. Preferredembodiments of the method consist of a DNA ligation operation, anamplification operation, and, optionally, a detection operation. The DNAligation operation circularizes a specially designed nucleic acid probemolecule. This step is dependent on hybridization of the probe to atarget sequence and forms circular probe molecules in proportion to theamount of target sequence present in a sample. The amplificationoperation is rolling circle replication of the circularized probe. Asingle round of amplification using rolling circle replication resultsin a large amplification of the circularized probe sequences, orders ofmagnitude greater than a single cycle of PCR replication and otheramplification techniques in which each cycle is limited to a doubling ofthe number of copies of a target sequence. Rolling circle amplificationcan also be performed independently of a ligation operation. By couplinga nucleic acid tag to a specific binding molecule, such as an antibody,amplification of the nucleic acid tag can be used to detect analytes ina sample. Optionally, an additional amplification operation can beperformed on the DNA produced by rolling circle replication.

Following amplification, the amplified probe sequences can be detectedand quantified using any of the conventional detection systems fornucleic acids such as detection of fluorescent labels, enzyme-linkeddetection systems, antibody-mediated label detection, and detection ofradioactive labels. Since the amplified product is directly proportionalto the amount of target sequence present in a sample, quantitativemeasurements reliably represent the amount of a target sequence in asample. Major advantages of this method are that the ligation operationcan be manipulated to obtain allelic discrimination, the amplificationoperation is isothermal, and signals are strictly quantitative becausethe amplification reaction is linear and is catalyzed by a highlyprocessive enzyme. In multiplex assays, the primer oligonucleotide usedfor DNA replication can be the same for all probes.

The disclosed method has two features that provide simple, quantitative,and consistent amplification and detection of a target nucleic acidsequence. First, target sequences are amplified via a small diagnosticprobe with an arbitrary primer binding sequence. This allows consistencyin the priming and replication reactions, even between probes havingvery different target sequences. Second, amplification takes place notin cycles, but in a continuous, isothermal replication: rolling circlereplication. This makes amplification less complicated and much moreconsistent in output.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an example of an open circle probe hybridized toa target sequence. The diagram shows the relationship between the targetsequence and the right and left target probes.

FIG. 2 is a diagram of an example of a gap oligonucleotide and an opencircle probe hybridized to a target sequence. The diagram shows therelationship between the target sequence, the gap oligonucleotide, andthe right and left target probes.

FIG. 3 is a diagram of an open circle probe hybridized and ligated to atarget sequence. The diagram shows how the open circle probe becomestopologically locked to the target sequence.

FIG. 4 is a diagram of rolling circle amplification of an open circleprobe topologically locked to the nucleic acid containing the targetsequence.

FIG. 5 is a diagram of an example of an open circle probe. Variousportions of the open circle probe are indicated by different fills.

FIG. 6 is a diagram of tandem sequence DNA (TS-DNA) and an address probedesigned to hybridize to the portion of the TS-DNA corresponding to partof the right and left target probes of the open circle probe and the gapoligonucleotide. The TS-DNA is SEQ ID NO:2 and the address probe is SEQID NO:3.

FIG. 7 is a diagram of the capture and detection of TS-DNA. Capture iseffected by hybridization of the TS-DNA to address probes attached to asolid-state detector. Detection is effected by hybridization ofsecondary detection probes to the captured TS-DNA. Portions of theTS-DNA corresponding to various portions of the open circle probe areindicated by different fills.

FIG. 8 is a diagram of an example of ligation-mediated rolling circlereplication followed by transcription (LM-RCT). Diagramed at the top isa gap oligonucleotide and an open circle probe, having a primercomplement portion and a promoter portion next to the right and lefttarget probe portions, respectively, hybridized to a target sequence.Diagramed at the bottom is the rolling circle replication producthybridized to unligated copies of the open circle probe and gapoligonucleotide. This hybridization forms the double-stranded substratefor transcription.

FIG. 9 is a diagram of an example of a multiplex antibody assayemploying open circle probes and LM-RCT for generation of an amplifiedsignal. Diagramed are three reporter antibodies, each with a differentoligonucleotide as a DNA tag. Diagramed at the bottom is amplificationof only that DNA tag coupled to a reporter antibody that bound.

FIG. 10 is a diagram of two schemes for multiplex detection of specificamplified nucleic acids. Diagramed at the top is hybridization ofdetection probes with different labels to amplified nucleic acids.Diagramed at the bottom is hybridization of amplified nucleic acid to asolid-state detector with address probes for the different possibleamplification products attached in a pattern.

FIGS. 11A and 11B are diagrams of an example of secondary DNA stranddisplacement. Diagramed at the top of FIG. 11A is a gap oligonucleotideand an open circle probe hybridized to a target sequence. Diagramed atthe bottom of FIG. 11A is the rolling circle replication producthybridized to secondary DNA strand displacement primers. Diagramed inFIG. 11B is secondary DNA strand displacement initiated from multipleprimers. FIG. 11B illustrates secondary DNA strand displacement carriedout simultaneously with rolling circle replication.

FIG. 12 is a diagram of an example of nested RCA using an unamplifiedfirst open circle probe as the target sequence. Diagramed at the top isa gap oligonucleotide and a first open circle probe hybridized to atarget sequence, and a secondary open circle probe hybridized to thefirst open circle probe. Diagramed at the bottom is the rolling circlereplication product of the secondary open circle probe.

FIG. 13 is a diagram of an example of strand displacement cascadeamplification. Diagramed is the synthesis and template relationships offour generations of TS-DNA. TS-DNA-1 is generated by rolling circlereplication primed by the rolling circle replication primer. TS-DNA-2and TS-DNA-4 are generated by secondary DNA strand displacement primedby a secondary DNA strand displacement primer (P2). TS-DNA-3 isgenerated by strand-displacing secondary DNA strand displacement primedby a tertiary DNA strand displacement primer (P1).

FIG. 14 is a diagram of an example of opposite strand amplification.Diagramed are five different stages of the reaction as DNA synthesisproceeds. TS-DNA-2 is generated by secondary DNA strand displacement ofTS-DNA primed by the secondary DNA strand displacement primer. Asrolling circle replication creates new TS-DNA sequence, the secondaryDNA strand displacement primer hybridizes to the newly synthesized DNAand primes synthesis of another copy of TS-DNA-2.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed composition and method make use of certain materials andprocedures which allow consistent and quantitative amplification anddetection of target nucleic acid sequences. These materials andprocedures are described in detail below.

Some major features of the disclosed method are:

-   1. The ligation operation can be manipulated to obtain allelic    discrimination, especially with the use of a gap-filling step.-   2. The amplification operation is isothermal.-   3. Signals can be strictly quantitative because in certain    embodiments of the amplification operation amplification is linear    and is catalyzed by a highly processive enzyme. In multiplex assays,    the primer used for DNA replication is the same for all probes.-   4. Modified nucleotides or other moieties may be incorporated during    DNA replication or transcription.-   5. The amplification product is a repetitive DNA molecule, and may    contain arbitrarily chosen tag sequences that are useful for    detection.

I. Materials

A. Open Circle Probes

An open circle probe (OCP) is a linear single-stranded DNA molecule,generally containing between 50 to 1000 nucleotides, preferably betweenabout 60 to 150 nucleotides, and most preferably between about 70 to 100nucleotides. The OCP has a 5′ phosphate group and a 3′ hydroxyl group.This allows the ends to be ligated using a DNA ligase, or extended in agap-filling operation. Portions of the OCP have specific functionsmaking the OCP useful for RCA and LM-RCA. These portions are referred toas the target probe portions, the primer complement portion, the spacerregion, the detection tag portions, the secondary target sequenceportions, the address tag portions, and the promoter portion (FIG. 5).The target probe portions and the primer complement portion are requiredelements of an open circle probe. The primer complement portion is partof the spacer region. Detection tag portions, secondary target sequenceportions, and promoter portions are optional and, when present, are partof the spacer region. Address tag portions are optional and, whenpresent, may be part of the spacer region. Generally, an open circleprobe is a single-stranded, linear DNA molecule comprising, from 5′ endto 3′ end, a 5′ phosphate group, a right target probe portion, a spacerregion, a left target probe portion, and a 3′ hydroxyl group, with aprimer complement portion present as part of the spacer region. Thosesegments of the spacer region that do not correspond to a specificportion of the OCP can be arbitrarily chosen sequences. It is preferredthat OCPs do not have any sequences that are self-complementary. It isconsidered that this condition is met if there are no complementaryregions greater than six nucleotides long without a mismatch or gap. Itis also preferred that OCPs containing a promoter portion do not haveany sequences that resemble a transcription terminator, such as a run ofeight or more thymidine nucleotides.

The open circle probe, when ligated and replicated, gives rise to a longDNA molecule containing multiple repeats of sequences complementary tothe open circle probe. This long DNA molecule is referred to herein astandem sequences DNA (TS-DNA). TS-DNA contains sequences complementaryto the target probe portions, the primer complement portion, the spacerregion, and, if present on the open circle probe, the detection tagportions, the secondary target sequence portions, the address tagportions, and the promoter portion. These sequences in the TS-DNA arereferred to as target sequences (which match the original targetsequence), primer sequences (which match the sequence of the rollingcircle replication primer), spacer sequences (complementary to thespacer region), detection tags, secondary target sequences, addresstags, and promoter sequences.

A particularly preferred embodiment is an open circle probe of 70 to 100nucleotides including a left target probe of 20 nucleotides and a righttarget probe of 20 nucleotides. The left target probe and right targetprobe hybridize to a target sequence leaving a gap of five nucleotides,which is filled by a single pentanucleotide gap oligonucleotide.

1. Target Probe Portions

There are two target probe portions on each OCP, one at each end of theOCP. The target probe portions can each be any length that supportsspecific and stable hybridization between the target probes and thetarget sequence. For this purpose, a length of 10 to 35 nucleotides foreach target probe portion is preferred, with target probe portions 15 to20 nucleotides long being most preferred. The target probe portion atthe 3′ end of the OCP is referred to as the left target probe, and thetarget probe portion at the 5′ end of the OCP is referred to as theright target probe. These target probe portions are also referred toherein as left and right target probes or left and right probes. Thetarget probe portions are complementary to a target nucleic acidsequence.

The target probe portions are complementary to the target sequence, suchthat upon hybridization the 5′ end of the right target probe portion andthe 3′ end of the left target probe portion are base-paired to adjacentnucleotides in the target sequence, with the objective that they serveas a substrate for ligation (FIG. 1). Optionally, the 5′ end and the 3′end of the target probe portions may hybridize in such a way that theyare separated by a gap space. In this case the 5′ end and the 3′ end ofthe OCP may only be ligated if one or more additional oligonucleotides,referred to as gap oligonucleotides, are used, or if the gap space isfilled during the ligation operation. The gap oligonucleotides hybridizeto the target sequence in the gap space to a form continuousprobe/target hybrid (FIG. 2). The gap space may be any length desiredbut is generally ten nucleotides or less. It is preferred that the gapspace is between about three to ten nucleotides in length, with a gapspace of four to eight nucleotides in length being most preferred.Alternatively, a gap space could be filled using a DNA polymerase duringthe ligation operation (see Example 3). When using such a gap-fillingoperation, a gap space of three to five nucleotides in length being mostpreferred.

2. Primer Complement Portion

The primer complement portion is part of the spacer region of an opencircle probe. The primer complement portion is complementary to therolling circle replication primer (RCRP). Each OCP should have a singleprimer complement portion. This allows rolling circle replication toinitiate at a single site on ligated OCPs. The primer complement portionand the cognate primer can have any desired sequence so long as they arecomplementary to each other. In general, the sequence of the primercomplement can be chosen such that it is not significantly similar toany other portion of the OCP. The primer complement portion can be anylength that supports specific and stable hybridization between theprimer complement portion and the primer. For this purpose, a length of10 to 35 nucleotides is preferred, with a primer complement portion 16to 20 nucleotides long being most preferred. The primer complementportion can be located anywhere within the spacer region of an OCP. Itis preferred that the primer complement portion is adjacent to the righttarget probe, with the right target probe portion and the primercomplement portion preferably separated by three to ten nucleotides, andmost preferably separated by six nucleotides. This location prevents thegeneration of any other spacer sequences, such as detection tags andsecondary target sequences, from unligated open circle probes during DNAreplication.

3. Detection Tag Portions

Detection tag portions are part of the spacer region of an open circleprobe. Detection tag portions have sequences matching the sequence ofthe complementary portion of detection probes. These detection tagportions, when amplified during rolling circle replication, result inTS-DNA having detection tag sequences that are complementary to thecomplementary portion of detection probes. If present, there may be one,two, three, or more than three detection tag portions on an OCP. It ispreferred that an OCP have two, three or four detection tag portions.Most preferably, an OCP will have three detection tag portions.Generally, it is preferred that an OCP have 60 detection tag portions orless. There is no fundamental limit to the number of detection tagportions that can be present on an OCP except the size of the OCP. Whenthere are multiple detection tag portions, they may have the samesequence or they may have different sequences, with each differentsequence complementary to a different detection probe. It is preferredthat an OCP contain detection tag portions that have the same sequencesuch that they are all complementary to a single detection probe. Thedetection tag portions can each be any length that supports specific andstable hybridization between the detection tags and the detection probe.For this purpose, a length of 10 to 35 nucleotides is preferred, with adetection tag portion 15 to 20 nucleotides long being most preferred.

4. Secondary Target Sequence Portions

Secondary target sequence portions are part of the spacer region of anopen circle probe. Secondary target sequence portions have sequencesmatching the sequence of target probes of a secondary open circle probe.These secondary target sequence portions, when amplified during rollingcircle replication, result in TS-DNA having secondary target sequencesthat are complementary to target probes of a secondary open circleprobe. If present, there may be one, two, or more than two secondarytarget sequence portions on an OCP. It is preferred that an OCP have oneor two secondary target sequence portions. Most preferably, an OCP willhave one secondary target sequence portion. Generally, it is preferredthat an OCP have 50 secondary target sequence portions or less. There isno fundamental limit to the number of secondary target sequence portionsthat can be present on an OCP except the size of the OCP. When there aremultiple secondary target sequence portions, they may have the samesequence or they may have different sequences, with each differentsequence complementary to a different secondary OCP. It is preferredthat an OCP contain secondary target sequence portions that have thesame sequence such that they are all complementary to a single targetprobe portion of a secondary OCP. The secondary target sequence portionscan each be any length that supports specific and stable hybridizationbetween the secondary target sequence and the target sequence probes ofits cognate OCP. For this purpose, a length of 20 to 70 nucleotides ispreferred, with a secondary target sequence portion 30 to 40 nucleotideslong being most preferred. As used herein, a secondary open circle probeis an open circle probe where the target probe portions match or arecomplementary to secondary target sequences in another open circle probeor an amplification target circle. It is contemplated that a secondaryopen circle probe can itself contain secondary target sequences thatmatch or are complementary to the target probe portions of anothersecondary open circle probe. Secondary open circle probes related toeach other in this manner are referred to herein as nested open circleprobes.

5. Address Tag Portion

The address tag portion is part of either the target probe portions orthe spacer region of an open circle probe. The address tag portion has asequence matching the sequence of the complementary portion of anaddress probe. This address tag portion, when amplified during rollingcircle replication, results in TS-DNA having address tag sequences thatare complementary to the complementary portion of address probes. Ifpresent, there may be one, or more than one, address tag portions on anOCP. It is preferred that an OCP have one or two address tag portions.Most preferably, an OCP will have one address tag portion. Generally, itis preferred that an OCP have 50 address tag portions or less. There isno fundamental limit to the number of address tag portions that can bepresent on an OCP except the size of the OCP. When there are multipleaddress tag portions, they may have the same sequence or they may havedifferent sequences, with each different sequence complementary to adifferent address probe. It is preferred that an OCP contain address tagportions that have the same sequence such that they are allcomplementary to a single address probe. Preferably, the address tagportion overlaps all or a portion of the target probe portions, and allof any intervening gap space (FIG. 6). Most preferably, the address tagportion overlaps all or a portion of both the left and right targetprobe portions. The address tag portion can be any length that supportsspecific and stable hybridization between the address tag and theaddress probe. For this purpose, a length between 10 and 35 nucleotideslong is preferred, with an address tag portion 15 to 20 nucleotides longbeing most preferred.

6. Promoter Portion

The promoter portion corresponds to the sequence of an RNA polymerasepromoter. A promoter portion can be included in an open circle probe sothat transcripts can be generated from TS-DNA. The sequence of anypromoter may be used, but simple promoters for RNA polymerases withoutcomplex requirements are preferred. It is also preferred that thepromoter is not recognized by any RNA polymerase that may be present inthe sample containing the target nucleic acid sequence. Preferably, thepromoter portion corresponds to the sequence of a T7 or SP6 RNApolymerase promoter. The T7 and SP6 RNA polymerases are highly specificfor particular promoter sequences. Other promoter sequences specific forRNA polymerases with this characteristic would also be preferred.Because promoter sequences are generally recognized by specific RNApolymerases, the cognate polymerase for the promoter portion of the OCPshould be used for transcriptional amplification. Numerous promotersequences are known and any promoter specific for a suitable RNApolymerase can be used. The promoter portion can be located anywherewithin the spacer region of an OCP and can be in either orientation.Preferably, the promoter portion is immediately adjacent to the lefttarget probe and is oriented to promote transcription toward the 3′ endof the open circle probe. This orientation results in transcripts thatare complementary to TS-DNA, allowing independent detection of TS-DNAand the transcripts, and prevents transcription from interfering withrolling circle replication.

B. Gap Oligonucleotides

Gap oligonucleotides are oligonucleotides that are complementary to allor a part of that portion of a target sequence which covers a gap spacebetween the ends of a hybridized open circle probe. An example of a gapoligonucleotide and its relationship to a target sequence and opencircle probe is shown in FIG. 2. Gap oligonucleotides have a phosphategroup at their 5′ ends and a hydroxyl group at their 3′ ends. Thisfacilitates ligation of gap oligonucleotides to open circle probes, orto other gap oligonucleotides. The gap space between the ends of ahybridized open circle probe can be filled with a single gapoligonucleotide, or it can be filled with multiple gap oligonucleotides.For example, two 3 nucleotide gap oligonucleotides can be used to fill asix nucleotide gap space, or a three nucleotide gap oligonucleotide anda four nucleotide gap oligonucleotide can be used to fill a sevennucleotide gap space. Gap oligonucleotides are particularly useful fordistinguishing between closely related target sequences. For example,multiple gap oligonucleotides can be used to amplify different allelicvariants of a target sequence. By placing the region of the targetsequence in which the variation occurs in the gap space formed by anopen circle probe, a single open circle probe can be used to amplifyeach of the individual variants by using an appropriate set of gapoligonucleotides.

C. Amplification Target Circles

An amplification target circle (ATC) is a circular single-stranded DNAmolecule, generally containing between 40 to 1000 nucleotides,preferably between about 50 to 150 nucleotides, and most preferablybetween about 50 to 100 nucleotides. Portions of ATCs have specificfunctions making the ATC useful for rolling circle amplification (RCA).These portions are referred to as the primer complement portion, thedetection tag portions, the secondary target sequence portions, theaddress tag portions, and the promoter portion. The primer complementportion is a required element of an amplification target circle.Detection tag portions, secondary target sequence portions, address tagportions, and promoter portions are optional. Generally, anamplification target circle is a single-stranded, circular DNA moleculecomprising a primer complement portion. Those segments of the ATC thatdo not correspond to a specific portion of the ATC can be arbitrarilychosen sequences. It is preferred that ATCs do not have any sequencesthat are self-complementary. It is considered that this condition is metif there are no complementary regions greater than six nucleotides longwithout a mismatch or gap. It is also preferred that ATCs containing apromoter portion do not have any sequences that resemble a transcriptionterminator, such as a run of eight or more thymidine nucleotides.Ligated open circle probes are a type of ATC, and as used herein theterm amplification target circle includes ligated open circle probes. AnATC can be used in the same manner as described herein for OCPs thathave been ligated.

An amplification target circle, when replicated, gives rise to a longDNA molecule containing multiple repeats of sequences complementary tothe amplification target circle. This long DNA molecule is referred toherein as tandem sequences DNA (TS-DNA). TS-DNA contains sequencescomplementary to the primer complement portion and, if present on theamplification target circle, the detection tag portions, the secondarytarget sequence portions, the address tag portions, and the promoterportion. These sequences in the TS-DNA are referred to as primersequences (which match the sequence of the rolling circle replicationprimer), spacer sequences (complementary to the spacer region),detection tags, secondary target sequences, address tags, and promotersequences. Amplification target circles are useful as tags for specificbinding molecules.

D. Rolling Circle Replication Primer

A rolling circle replication primer (RCRP) is an oligonucleotide havingsequence complementary to the primer complement portion of an OCP orATC. This sequence is referred to as the complementary portion of theRCRP. The complementary portion of a RCRP and the cognate primercomplement portion can have any desired sequence so long as they arecomplementary to each other. In general, the sequence of the RCRP can bechosen such that it is not significantly complementary to any otherportion of the OCP or ATC. The complementary portion of a rolling circlereplication primer can be any length that supports specific and stablehybridization between the primer and the primer complement portion.Generally this is 10 to 35 nucleotides long, but is preferably 16 to 20nucleotides long.

It is preferred that rolling circle replication primers also containadditional sequence at the 5′ end of the RCRP that is not complementaryto any part of the OCP or ATC. This sequence is referred to as thenon-complementary portion of the RCRP. The non-complementary portion ofthe RCRP, if present, serves to facilitate strand displacement duringDNA replication. The non-complementary portion of a RCRP may be anylength, but is generally 1 to 100 nucleotides long, and preferably 4 to8 nucleotides long. The rolling circle replication primer may alsoinclude modified nucleotides to make it resistant to exonucleasedigestion. For example, the primer can have three or fourphosphorothioate linkages between nucleotides at the 5′ end of theprimer. Such nuclease resistant primers allow selective degradation ofexcess unligated OCP and gap oligonucleotides that might otherwiseinterfere with hybridization of detection probes, address probes, andsecondary OCPs to the amplified nucleic acid. A rolling circlereplication primer can be used as the tertiary DNA strand displacementprimer in strand displacement cascade amplification.

E. Detection Labels

To aid in detection and quantitation of nucleic acids amplified usingRCA and RCT, detection labels can be directly incorporated intoamplified nucleic acids or can be coupled to detection molecules. Asused herein, a detection label is any molecule that can be associatedwith amplified nucleic acid, directly or indirectly, and which resultsin a measurable, detectable signal, either directly or indirectly. Manysuch labels for incorporation into nucleic acids or coupling to nucleicacid or antibody probes are known to those of skill in the art. Examplesof detection labels suitable for use in RCA and RCT are radioactiveisotopes, fluorescent molecules, phosphorescent molecules, enzymes,antibodies, and ligands.

Examples of suitable fluorescent labels include fluorescein,5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, andrhodamine. Preferred fluorescent labels are fluorescein(5-carboxyfluorescein-N-hydroxysuccinimide ester) and rhodamine(5,6-tetramethyl rhodamine). These can be obtained from a variety ofcommercial sources, including Molecular Probes, Eugene, Oreg. andResearch Organics, Cleveland, Ohio.

Labeled nucleotides are preferred form of detection label since they canbe directly incorporated into the products of RCA and RCT duringsynthesis. Examples of detection labels that can be incorporated intoamplified DNA or RNA include nucleotide analogs such as BrdUrd (Hoy andSchimke, Mutation Research 290:217-230 (1993)), BrUTP (Wansick et al.,J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin(Langer et al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or withsuitable haptens such as digoxygenin (Kerkhof, Anal. Biochem.205:359-364 (1992)). Suitable fluorescence-labeled nucleotides areFluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yuet al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotideanalog detection label for DNA is BrdUrd (BUDR triphosphate, Sigma), anda preferred nucleotide analog detection label for RNA isBiotin-16-uridine-5′-triphosphate (Biotin-16-dUTP, BoehringherMannheim).

Detection labels that are incorporated into amplified nucleic acid, suchas biotin, can be subsequently detected using sensitive methodswell-known in the art. For example, biotin can be detected usingstreptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which isbound to the biotin and subsequently detected by chemiluminescence ofsuitable substrates (for example, chemiluminescent substrate CSPD:disodium, 3-(4-methoxyspiro-[1,2,-dioxetane-3-2′-(5′-chloro)tricyclo[3,3,1,1^(3,7)]decane]-4-yl)phenyl phosphate; Tropix, Inc.).

A preferred detection label for use in detection of amplified RNA isacridinium-ester-labeled DNA probe (GenProbe, Inc., as described byArnold et al., Clinical Chemistry 35:1588-1594 (1989)). Anacridinium-ester-labeled detection probe permits the detection ofamplified RNA without washing because unhybridized probe can bedestroyed with alkali (Arnold et al. (1989)).

Molecules that combine two or more of these detection labels are alsoconsidered detection labels. Any of the known detection labels can beused with the disclosed probes, tags, and method to label and detectnucleic acid amplified using the disclosed method. Methods for detectingand measuring signals generated by detection labels are also known tothose of skill in the art. For example, radioactive isotopes can bedetected by scintillation counting or direct visualization; fluorescentmolecules can be detected with fluorescent spectrophotometers;phosphorescent molecules can be detected with a spectrophotometer ordirectly visualized with a camera; enzymes can be detected by detectionor visualization of the product of a reaction catalyzed by the enzyme;antibodies can be detected by detecting a secondary detection labelcoupled to the antibody. Such methods can be used directly in thedisclosed method of amplification and detection. As used herein,detection molecules are molecules which interact with amplified nucleicacid and to which one or more detection labels are coupled.

F. Detection Probes

Detection probes are labeled oligonucleotides having sequencecomplementary to detection tags on TS-DNA or transcripts of TS-DNA. Thecomplementary portion of a detection probe can be any length thatsupports specific and stable hybridization between the detection probeand the detection tag. For this purpose, a length of 10 to 35nucleotides is preferred, with a complementary portion of a detectionprobe 16 to 20 nucleotides long being most preferred. Detection probescan contain any of the detection labels described above. Preferredlabels are biotin and fluorescent molecules. A particularly preferreddetection probe is a molecular beacon. Molecular beacons are detectionprobes labeled with fluorescent moieties where the fluorescent moietiesfluoresce only when the detection probe is hybridized. The use of suchprobes eliminates the need for removal of unhybridized probes prior tolabel detection because the unhybridized detection probes will notproduce a signal. This is especially useful in multiplex assays.

G. Address Probes

An address probe is an oligonucleotide having a sequence complementaryto address tags on TS-DNA or transcripts of TS-DNA. The complementaryportion of an address probe can be any length that supports specific andstable hybridization between the address probe and the address tag. Forthis purpose, a length of 10 to 35 nucleotides is preferred, with acomplementary portion of an address probe 12 to 18 nucleotides longbeing most preferred. Preferably, the complementary portion of anaddress probe is complementary to all or a portion of the target probeportions of an OCP. Most preferably, the complementary portion of anaddress probe is complementary to a portion of either or both of theleft and right target probe portions of an OCP and all or a part of anygap oligonucleotides or gap sequence created in a gap-filling operation(see FIG. 6). Address probe can contain a single complementary portionor multiple complementary portions. Preferably, address probes arecoupled, either directly or via a spacer molecule, to a solid-statesupport. Such a combination of address probe and solid-state support area preferred form of solid-state detector.

H. DNA Strand Displacement Primers

Primers used for secondary DNA strand displacement are referred toherein as DNA strand displacement primers. One form of DNA stranddisplacement primer, referred to herein as a secondary DNA stranddisplacement primer, is an oligonucleotide having sequence matching partof the sequence of an OCP or ATC. This sequence is referred to as thematching portion of the secondary DNA strand displacement primer. Thismatching portion of a secondary DNA strand displacement primer iscomplementary to sequences in TS-DNA. The matching portion of asecondary DNA strand displacement primer may be complementary to anysequence in TS-DNA. However, it is preferred that it not becomplementary TS-DNA sequence matching either the rolling circlereplication primer or a tertiary DNA strand displacement primer, if oneis being used. This prevents hybridization of the primers to each other.The matching portion of a secondary DNA strand displacement primer maybe complementary to all or a portion of the target sequence. In thiscase, it is preferred that the 3′ end nucleotides of the secondary DNAstrand displacement primer are complementary to the gap sequence in thetarget sequence. It is most preferred that nucleotide at the 3′ end ofthe secondary DNA strand displacement primer falls complementary to thelast nucleotide in the gap sequence of the target sequence, that is, the5′ nucleotide in the gap sequence of the target sequence. The matchingportion of a secondary DNA strand displacement primer can be any lengththat supports specific and stable hybridization between the primer andits complement. Generally this is 12 to 35 nucleotides long, but ispreferably 18 to 25 nucleotides long.

It is preferred that secondary DNA strand displacement primers alsocontain additional sequence at their 5′ end that does not match any partof the OCP or ATC. This sequence is referred to as the non-matchingportion of the secondary DNA strand displacement primer. Thenon-matching portion of the secondary DNA strand displacement primer, ifpresent, serves to facilitate strand displacement during DNAreplication. The non-matching portion of a secondary DNA stranddisplacement primer may be any length, but is generally 1 to 100nucleotides long, and preferably 4 to 8 nucleotides long.

Another form of DNA strand displacement primer, referred to herein as atertiary DNA strand displacement primer, is an oligonucleotide havingsequence complementary to part of the sequence of an OCP or ATC. Thissequence is referred to as the complementary portion of the tertiary DNAstrand displacement primer. This complementary portion of the tertiaryDNA strand displacement primer matches sequences in TS-DNA. Thecomplementary portion of a tertiary DNA strand displacement primer maybe complementary to any sequence in the OCP or ATC. However, it ispreferred that it not be complementary OCP or ATC sequence matching thesecondary DNA strand displacement primer. This prevents hybridization ofthe primers to each other. Preferably, the complementary portion of thetertiary DNA strand displacement primer has sequence complementary to aportion of the spacer portion of an OCP. The complementary portion of atertiary DNA strand displacement primer can be any length that supportsspecific and stable hybridization between the primer and its complement.Generally this is 12 to 35 nucleotides long, but is preferably 18 to 25nucleotides long. It is preferred that tertiary DNA strand displacementprimers also contain additional sequence at their 5′ end that is notcomplementary to any part of the OCP or ATC. This sequence is referredto as the non-complementary portion of the tertiary DNA stranddisplacement primer. The non-complementary portion of the tertiary DNAstrand displacement primer, if present, serves to facilitate stranddisplacement during DNA replication. The non-complementary portion of atertiary DNA strand displacement primer may be any length, but isgenerally 1 to 100 nucleotides long, and preferably 4 to 8 nucleotideslong. A rolling circle replication primer is a preferred form oftertiary DNA strand displacement primer.

DNA strand displacement primers may also include modified nucleotides tomake them resistant to exonuclease digestion. For example, the primercan have three or four phosphorothioate linkages between nucleotides atthe 5′ end of the primer. Such nuclease resistant primers allowselective degradation of excess unligated OCP and gap oligonucleotidesthat might otherwise interfere with hybridization of detection probes,address probes, and secondary OCPs to the amplified nucleic acid. DNAstrand displacement primers can be used for secondary DNA stranddisplacement and strand displacement cascade amplification, bothdescribed below.

I. Peptide Nucleic Acid Clamps

Peptide nucleic acids (PNA) are a modified form of nucleic acid having apeptide backbone. Peptide nucleic acids form extremely stable hybridswith DNA (Hanvey et al., Science 258:1481-1485 (1992); Nielsen et al.,Anticancer Drug Des. 8:53-63 (1993)), and have been used as specificblockers of PCR reactions (Orum et al., Nucleic Acids Res., 21:5332-5336(1993)). PNA clamps are peptide nucleic acids complementary to sequencesin both the left target probe portion and right target probe portion ofan OCP, but not to the sequence of any gap oligonucleotides or filledgap space in the ligated OCP. Thus, a PNA clamp can hybridize only tothe ligated junction of OCPs that have been illegitimately ligated, thatis, ligated in a non-target-directed manner. The PNA clamp can be anylength that supports specific and stable hybridization between the clampand its complement. Generally this is 7 to 12 nucleotides long, but ispreferably 8 to 10 nucleotides long. PNA clamps can be used to reducebackground signals in rolling circle amplifications by preventingreplication of illegitimately ligated OCPs.

J. Oligonucleotide Synthesis

Open circle probes, gap oligonucleotides, rolling circle replicationprimers, detection probes, address probes, amplification target circles,DNA strand displacement primers, and any other oligonucleotides can besynthesized using established oligonucleotide synthesis methods. Methodsto produce or synthesize oligonucleotides are well known in the art.Such methods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

Many of the oligonucleotides described herein are designed to becomplementary to certain portions of other oligonucleotides or nucleicacids such that stable hybrids can be formed between them. The stabilityof these hybrids can be calculated using known methods such as thosedescribed in Lesnick and Freier, Biochemistry 34:10807-10815 (1995),McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik et al.,Nucleic Acids Res. 18:6409-6412 (1990).

K. Solid-State Detectors

Solid-state detectors are solid-state substrates or supports to whichaddress probes or detection molecules have been coupled. A preferredform of solid-state detector is an array detector. An array detector isa solid-state detector to which multiple different address probes ordetection molecules have been coupled in an array, grid, or otherorganized pattern.

Solid-state substrates for use in solid-state detectors can include anysolid material to which oligonucleotides can be coupled. This includesmaterials such as acrylamide, cellulose, nitrocellulose, glass,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, fibers, wovenfibers, shaped polymers, particles and microparticles. A preferred formfor a solid-state substrate is a microtiter dish. The most preferredform of microtiter dish is the standard 96-well type.

Address probes immobilized on a solid-state substrate allow capture ofthe products of RCA and RCT on a solid-state detector. Such captureprovides a convenient means of washing away reaction components thatmight interfere with subsequent detection steps. By attaching differentaddress probes to different regions of a solid-state detector, differentRCA or RCT products can be captured at different, and thereforediagnostic, locations on the solid-state detector. For example, in amicrotiter plate multiplex assay, address probes specific for up to 96different TS-DNAs (each amplified via a different target sequence) canbe immobilized on a microtiter plate, each in a different well. Captureand detection will occur only in those wells corresponding to TS-DNAsfor which the corresponding target sequences were present in a sample.

Methods for immobilization of oligonucleotides to solid-state substratesare well established. Oligonucleotides, including address probes anddetection probes, can be coupled to substrates using establishedcoupling methods. For example, suitable attachment methods are describedby Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), andKhrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method forimmobilization of 3′-amine oligonucleotides on casein-coated slides isdescribed by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383(1995). A preferred method of attaching oligonucleotides to solid-statesubstrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465(1994).

Some solid-state detectors useful in RCA and RCT assays have detectionantibodies attached to a solid-state substrate. Such antibodies can bespecific for a molecule of interest. Captured molecules of interest canthen be detected by binding of a second, reporter antibody, followed byRCA or RCT. Such a use of antibodies in a solid-state detector allowsRCA assays to be developed for the detection of any molecule for whichantibodies can be generated. Methods for immobilizing antibodies tosolid-state substrates are well established. Immobilization can beaccomplished by attachment, for example, to aminated surfaces,carboxylated surfaces or hydroxylated surfaces using standardimmobilization chemistries. Examples of attachment agents are cyanogenbromide, succinimide, aldehydes, tosyl chloride, avidin-biotin,photocrosslinkable agents, epoxides and maleimides. A preferredattachment agent is glutaraldehyde. These and other attachment agents,as well as methods for their use in attachment, are described in Proteinimmobilization: fundamentals and applications, Richard F. Taylor, ed.(M. Dekker, New York, 1991), and Johnstone and Thorpe, ImmunochemistryIn Practice (Blackwell Scientific Publications, Oxford, England, 1987)pages 209-216 and 241-242. Antibodies can be attached to a substrate bychemically cross-linking a free amino group on the antibody to reactiveside groups present within the solid-state substrate. For example,antibodies may be chemically cross-linked to a substrate that containsfree amino or carboxyl groups using glutaraldehyde or carbodiimides ascross-linker agents. In this method, aqueous solutions containing freeantibodies are incubated with the solid-state substrate in the presenceof glutaraldehyde or carbodiimide. For crosslinking with glutaraldehydethe reactants can be incubated with 2% glutaraldehyde by volume in abuffered solution such as 0.1 M sodium cacodylate at pH 7.4. Otherstandard immobilization chemistries are known by those of skill in theart.

L. Reporter Binding Agents

A reporter binding agent is a specific binding molecule coupled ortethered to an oligonucleotide. The specific binding molecule isreferred to as the affinity portion of the reporter binding agent andthe oligonucleotide is referred to as the oligonucleotide portion of thereporter binding agent. As used herein, a specific binding molecule is amolecule that interacts specifically with a particular molecule ormoiety. Antibodies, either member of a receptor/ligand pair, and othermolecules with specific binding affinities are examples of specificbinding molecules, useful as the affinity portion of a reporter bindingmolecule. By tethering an amplification target circle or coupling atarget sequence to such specific binding molecules, binding of aspecific binding molecule to its specific target can be detected byamplifying the ATC or target sequence with rolling circle amplification.This amplification allows sensitive detection of a very small number ofbound specific binding molecules.

In one embodiment, the oligonucleotide portion of a reporter bindingagent includes a sequence, referred to as a target sequence, that servesas a target sequence for an OCP. The sequence of the target sequence canbe arbitrarily chosen. In a multiplex assay using multiple reporterbinding agents, it is preferred that the target sequence for eachreporter binding agent be substantially different to limit thepossibility of non-specific target detection. Alternatively, it may bedesirable in some multiplex assays, to use target sequences with relatedsequences. By using different, unique gap oligonucleotides to filldifferent gap spaces, such assays can use one or a few OCPs to amplifyand detect a larger number of target sequences. The oligonucleotideportion can be coupled to the affinity portion by any of severalestablished coupling reactions. For example, Hendrickson et al., NucleicAcids Res., 23(3):522-529 (1995) describes a suitable method forcoupling oligonucleotides to antibodies.

In another embodiment, the oligonucleotide portion of a reporter bindingagent can include an amplification target circle which serves as atemplate for rolling circle replication. In a multiplex assay usingmultiple reporter binding agents, it is preferred that address tagportions and detection tag portions of the ATC comprising theoligonucleotide portion of each reporter binding agent be substantiallydifferent to unique detection of each reporter binding agent. It isdesirable, however, to use the same primer complement portion in all ofthe ATCs used in a multiplex assay. The ATC is tethered to the specificbinding molecule by looping the ATC around a tether loop. This allowsthe ATC to rotate freely during rolling circle replication whileremaining coupled to the affinity portion. The tether loop can be anymaterial that can form a loop and be coupled to a specific bindingmolecule. Linear polymers are a preferred material for tether loops.

A preferred method of producing a reporter binding agent with a tetheredATC is to form the tether loop by ligating the ends of oligonucleotidescoupled to a specific binding molecule around an ATC. Oligonucleotidescan be coupled to specific binding molecules using known techniques. Forexample, Hendrickson et al. (1995), describes a suitable method forcoupling oligonucleotides to antibodies. This method is generally usefulfor coupling oligonucleotides to any protein. To allow ligation,oligonucleotides comprising the two halves of the tether loop should becoupled to the specific binding molecule in opposite orientations suchthat the free end of one is the 5′ end and the free end of the other isthe 3′ end. Ligation of the ends of the tether oligonucleotides can bemediated by hybridization of the ends of the tether oligonucleotides toadjacent sequences in the ATC to be tethered. In this way, the ends ofthe tether oligonucleotides are analogous to the target probe portionsof an open circle probe, with the ATC containing the target sequence.

Another preferred method of producing a reporter binding agent with atethered ATC is to ligate an open circle probe while hybridized to anoligonucleotide tether loop on a specific binding molecule. This isanalogous to the ligation operation of LM-RCA. In this case, the targetsequence is part of an oligonucleotide with both ends coupled to aspecific binding molecule. In this method, both ends of a single tetheroligonucleotide are coupled to a specific binding molecule. This can beaccomplished using known coupling techniques as described above.

The ends of tether loops can be coupled to any specific binding moleculewith functional groups that can be derivatized with suitable activatinggroups. When the specific binding molecule is a protein, or a moleculewith similar functional groups, coupling of tether ends can beaccomplished using known methods of protein attachment. Many suchmethods are described in Protein immobilization: fundamentals andapplications Richard F. Taylor, ed. (M. Dekker, New York, 1991).

Antibodies useful as the affinity portion of reporter binding agents,can be obtained commercially or produced using well established methods.For example, Johnstone and Thorpe, on pages 30-85, describe generalmethods useful for producing both polyclonal and monoclonal antibodies.The entire book describes many general techniques and principles for theuse of antibodies in assay systems.

M. DNA Ligases

Any DNA ligase is suitable for use in the disclosed amplificationmethod. Preferred ligases are those that preferentially formphosphodiester bonds at nicks in double-stranded DNA. That is, ligasesthat fail to ligate the free ends of single-stranded DNA at asignificant rate are preferred. Thermostable ligases are especiallypreferred. Many suitable ligases are known, such as T4 DNA ligase (Daviset al., Advanced Bacterial Genetics—A Manual for Genetic Engineering(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1980)), E.coli DNA ligase (Panasnko et al., J. Biol. Chem. 253:4590-4592 (1978)),AMPLIGASE® (Kalin et al., Mutat. Res., 283(2):119-123 (1992); Winn-Deenet al., Mol Cell Probes (England) 7(3):179-186 (1993)), Taq DNA ligase(Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991), Thermusthermophilus DNA ligase (Abbott Laboratories), Thermus scotoductus DNAligase and Rhodothermus marinus DNA ligase (Thorbjarnardottir et al.,Gene 151:177-180 (1995)). T4 DNA ligase is preferred for ligationsinvolving RNA target sequences due to its ability to ligate DNA endsinvolved in DNA:RNA hybrids (Hsuih et al., Quantitative detection of HCVRNA using novel ligation-dependent polymerase chain reaction, AmericanAssociation for the Study of Liver Diseases (Chicago, Ill., Nov. 3-7,1995)).

The frequency of non-target-directed ligation catalyzed by a ligase canbe determined as follows. LM-RCA is performed with an open circle probeand a gap oligonucleotide in the presence of a target sequence.Non-targeted-directed ligation products can then be detected by using anaddress probe specific for the open circle probe ligated without the gapoligonucleotide to capture TS-DNA from such ligated probes. Targetdirected ligation products can be detected by using an address probespecific for the open circle probe ligated with the gap oligonucleotide.By using a solid-state detector with regions containing each of theseaddress probes, both target directed and non-target-directed ligationproducts can be detected and quantitated. The ratio of target-directedand non-target-directed TS-DNA produced provides a measure of thespecificity of the ligation operation. Target-directed ligation can alsobe assessed as discussed in Barany (1991).

N. DNA Polymerases

DNA polymerases useful in the rolling circle replication step of RCAmust perform rolling circle replication of primed single-strandedcircles. Such polymerases are referred to herein as rolling circle DNApolymerases. For rolling circle replication, it is preferred that a DNApolymerase be capable of displacing the strand complementary to thetemplate strand, termed strand displacement, and lack a 5′ to 3′exonuclease activity. Strand displacement is necessary to result insynthesis of multiple tandem copies of the ligated OCP. A 5′ to 3′exonuclease activity, if present, might result in the destruction of thesynthesized strand. It is also preferred that DNA polymerases for use inthe disclosed method are highly processive. The suitability of a DNApolymerase for use in the disclosed method can be readily determined byassessing its ability to carry out rolling circle replication. Preferredrolling circle DNA polymerases are bacteriophage φ29 DNA polymerase(U.S. Pat. Nos. 5,198,543 and 5,001,050 to Blanco et al.), phage M2 DNApolymerase (Matsumoto et al., Gene 84:247 (1989)), phage φPRD1 DNApolymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)),VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)),Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem.45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19(1991)), PRD1 DNA polymerase (Zhu and Ito, Biochim. Biophys. Acta.1219:267-276 (1994)), and T4 DNA polymerase holoenzyme (Kaboord andBenkovic, Curr. Biol. 5:149-157 (1995)). φ29 DNA polymerase is mostpreferred.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as helicase. It is considered that any DNApolymerase that can perform rolling circle replication in the presenceof a strand displacement factor is suitable for use in the disclosedmethod, even if the DNA polymerase does not perform rolling circlereplication in the absence of such a factor. Strand displacement factorsuseful in RCA include BMRF1 polymerase accessory subunit (Tsurumi etal., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-bindingprotein (Zijderveld and van der Vliet, J. Virology 68(2): 1158-1164(1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J.Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad.Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA bindingproteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)),and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635(1992)).

The ability of a polymerase to carry out rolling circle replication canbe determined by using the polymerase in a rolling circle replicationassay such as those described in Fire and Xu, Proc. Natl. Acad. Sci. USA92:4641-4645 (1995) and in Example 1.

Another type of DNA polymerase can be used if a gap-filling synthesisstep is used, such as in gap-filling LM-RCA (see Example 3). When usinga DNA polymerase to fill gaps, strand displacement by the DNA polymeraseis undesirable. Such DNA polymerases are referred to herein asgap-filling DNA polymerases. Unless otherwise indicated, a DNApolymerase referred to herein without specifying it as a rolling circleDNA polymerase or a gap-filling DNA polymerase, is understood to be arolling circle DNA polymerase and not a gap-filling DNA polymerase.Preferred gap-filling DNA polymerases are T7 DNA polymerase (Studier etal., Methods Enzymol. 185:60-89 (1990)), DEEP VENT® DNA polymerase (NewEngland Biolabs, Beverly, Mass.), and T4 DNA polymerase (Kunkel et al.,Methods Enzymol. 154:367-382 (1987)). An especially preferred type ofgap-filling DNA polymerase is the Thermus flavus DNA polymerase (MBR,Milwaukee, Wis.). The most preferred gap-filling DNA polymerase is theStoffel fragment of Taq DNA polymerase (Lawyer et al., PCR Methods Appl.2(4):275-287 (1993), King et al., J. Biol. Chem. 269(18):13061-13064(1994)).

The ability of a polymerase to fill gaps can be determined by performinggap-filling LM-RCA. Gap-filling LM-RCA is performed with an open circleprobe that forms a gap space when hybridized to the target sequence.Ligation can only occur when the gap space is filled by the DNApolymerase. If gap-filling occurs, TS-DNA can be detected, otherwise itcan be concluded that the DNA polymerase, or the reaction conditions, isnot useful as a gap-filling DNA polymerase.

O. RNA Polymerases

Any RNA polymerase which can carry out transcription in vitro and forwhich promoter sequences have been identified can be used in thedisclosed rolling circle transcription method. Stable RNA polymeraseswithout complex requirements are preferred. Most preferred are T7 RNApolymerase (Davanloo et al., Proc. Natl. Acad. Sci. USA 81:2035-2039(1984)) and SP6 RNA polymerase (Butler and Chamberlin, J. Biol. Chem.257:5772-5778 (1982)) which are highly specific for particular promotersequences (Schenbom and Meirendorf, Nucleic Acids Research 13:6223-6236(1985)). Other RNA polymerases with this characteristic are alsopreferred. Because promoter sequences are generally recognized byspecific RNA polymerases, the OCP or ATC should contain a promotersequence recognized by the RNA polymerase that is used. Numerouspromoter sequences are known and any suitable RNA polymerase having anidentified promoter sequence can be used. Promoter sequences for RNApolymerases can be identified using established techniques.

The materials described above can be packaged together in any suitablecombination as a kit useful for performing the disclosed method.

II. Method

The disclosed rolling circle amplification (RCA) method involvesreplication of circular single-stranded DNA molecules. In RCA, a rollingcircle replication primer hybridizes to circular OCP or ATC moleculesfollowed by rolling circle replication of the OCP or ATC molecules usinga strand-displacing DNA polymerase. Amplification takes place duringrolling circle replication in a single reaction cycle. Rolling circlereplication results in large DNA molecules containing tandem repeats ofthe OCP or ATC sequence. This DNA molecule is referred to as a tandemsequence DNA (TS-DNA).

A preferred embodiment, ligation-mediated rolling circle amplification(LM-RCA) method involves a ligation operation prior to replication. Inthe ligation operation, an OCP hybridizes to its cognate target nucleicacid sequence, if present, followed by ligation of the ends of thehybridized OCP to form a covalently closed, single-stranded OCP. Afterligation, a rolling circle replication primer hybridizes to OCPmolecules followed by rolling circle replication of the circular OCPmolecules using a strand-displacing DNA polymerase. Generally, LM-RCAcomprises

(a) mixing an open circle probe (OCP) with a target sample, resulting inan OCP-target sample mixture, and incubating the OCP-target samplemixture under conditions promoting hybridization between the open circleprobe and a target sequence,

(b) mixing ligase with the OCP-target sample mixture, resulting in aligation mixture, and incubating the ligation mixture under conditionspromoting ligation of the open circle probe to form an amplificationtarget circle (ATC),

(c) mixing a rolling circle replication primer (RCRP) with the ligationmixture, resulting in a primer-ATC mixture, and incubating theprimer-ATC mixture under conditions that promote hybridization betweenthe amplification target circle and the rolling circle replicationprimer,

(d) mixing DNA polymerase with the primer-ATC mixture, resulting in apolymerase-ATC mixture, and incubating the polymerase-ATC mixture underconditions promoting replication of the amplification target circle,where replication of the amplification target circle results information of tandem sequence DNA (TS-DNA).

The open circle probe is a single-stranded, linear DNA moleculecomprising, from 5′ end to 3′ end, a 5′ phosphate group, a right targetprobe portion, a primer complement portion, a spacer region, a lefttarget probe portion, and a 3′ hydroxyl group, wherein the left targetprobe portion is complementary to the 5′ region of a target sequence andthe right target probe portion is complementary to the 3′ region of thetarget sequence.

The left and right target probe portions hybridize to the two ends ofthe target nucleic acid sequence, with or without a central gap to befilled by one or more gap oligonucleotides. Generally, LM-RCA using gapoligonucleotides can be performed by, in an LM-RCA reaction, (1) using atarget sequence with a central region located between a 5′ region and a3′ region, and an OCP where neither the left target probe portion of theopen circle probe nor the right target probe portion of the open circleprobe is complementary to the central region of the target sequence, and(2) mixing one or more gap oligonucleotides with the target sample, suchthat the OCP-target sample mixture contains the open circle probe, theone or more gap oligonucleotides, and the target sample, where each gapoligonucleotide consists of a single-stranded, linear DNA moleculecomprising a 5′ phosphate group and a 3′ hydroxyl group, where each gapoligonucleotide is complementary all or a portion of the central regionof the target sequence.

A. The Ligation Operation

An open circle probe, optionally in the presence of one or more gapoligonucleotides, is incubated with a sample containing DNA, RNA, orboth, under suitable hybridization conditions, and then ligated to forma covalently closed circle. The ligated open circle probe is a form ofamplification target circle. This operation is similar to ligation ofpadlock probes described by Nilsson et al., Science, 265:2085-2088(1994). The ligation operation allows subsequent amplification to bedependent on the presence of a target sequence. Suitable ligases for theligation operation are described above. Ligation conditions aregenerally known. Most ligases require Mg⁺⁺. There are two main types ofligases, those that are ATP-dependent and those that are NAD-dependent.ATP or NAD, depending on the type of ligase, should be present duringligation.

The ligase and ligation conditions can be optimized to limit thefrequency of ligation of single-stranded termini. Such ligation eventsdo not depend on the presence of a target sequence. In the case ofAMPLIGASE®-catalyzed ligation, which takes place at 60° C., it isestimated that no more than 1 in 1,000,000 molecules withsingle-stranded DNA termini will be ligated. This is based on the levelof non-specific amplification seen with this ligase in the ligase chainreaction. Any higher nonspecific ligation frequency would causeenormously high background amplification in the ligase chain reaction.Using this estimate, an approximate frequency for the generation ofnon-specifically ligated open circles with a correctly placed gapoligonucleotide in at the ligation junction can be calculated. Since twoligation events are involved, the frequency of such events usingAMPLIGASE® should be the square of 1 in 1,000,000, or 1 in 1×10¹². Thenumber of probes used in a typical ligation reaction of 50 μl is 2×10¹².Thus, the number of non-specifically ligated circles containing acorrect gap oligonucleotide would be expected to be about 2 perreaction.

When RNA is to be detected, it is preferred that a reverse transcriptionoperation be performed to make a DNA target sequence. An example of theuse of such an operation is described in Example 4. Alternatively, anRNA target sequence can be detected directly by using a ligase that canperform ligation on a DNA:RNA hybrid substrate. A preferred ligase forthis is T4 DNA ligase.

B. The Replication Operation

The circular open circle probes formed by specific ligation andamplification target circles serve as substrates for a rolling circlereplication. This reaction requires the addition of two reagents: (a) arolling circle replication primer, which is complementary to the primercomplement portion of the OCP or ATC, and (b) a rolling circle DNApolymerase. The DNA polymerase catalyzes primer extension and stranddisplacement in a processive rolling circle polymerization reaction thatproceeds as long as desired, generating a molecule of up to 100,000nucleotides or larger that contains up to approximately 1000 tandemcopies of a sequence complementary to the amplification target circle oropen circle probe (FIG. 4). This tandem sequence DNA (TS-DNA) consistsof, in the case of OCPs, alternating target sequence and spacersequence. Note that the spacer sequence of the TS-DNA is the complementof the sequence between the left target probe and the right target probein the original open circle probe. A preferred rolling circle DNApolymerase is the DNA polymerase of the bacteriophage φ29.

During rolling circle replication one may additionally includeradioactive, or modified nucleotides such as bromodeoxyuridinetriphosphate, in order to label the DNA generated in the reaction.Alternatively, one may include suitable precursors that provide abinding moiety such as biotinylated nucleotides (Langer et al. (1981)).

C. Modifications and Additional Operations

1. Detection of Amplification Products

Current detection technology makes a second cycle of RCA unnecessary inmany cases. Thus, one may proceed to detect the products of the firstcycle of RCA directly. Detection may be accomplished by primary labelingor by secondary labeling, as described below.

(a) Primary Labeling

Primary labeling consists of incorporating labeled moieties, such asfluorescent nucleotides, biotinylated nucleotides,digoxygenin-containing nucleotides, or bromodeoxyuridine, during rollingcircle replication in RCA, or during transcription in RCT. For example,one may incorporate cyanine dye UTP analogs (Yu et al. (1994)) at afrequency of 4 analogs for every 100 nucleotides. A preferred method fordetecting nucleic acid amplified in situ is to label the DNA duringamplification with BrdUrd, followed by binding of the incorporated BUDRwith a biotinylated anti-BUDR antibody (Zymed Labs, San Francisco,Calif.), followed by binding of the biotin moieties withStreptavidin-Peroxidase (Life Sciences, Inc.), and finally developmentof fluorescence with Fluorescein-tyramide (DuPont de Nemours & Co.,Medical Products Dept.).

(b) Secondary Labeling with Detection Probes

Secondary labeling consists of using suitable molecular probes, referredto as detection probes, to detect the amplified DNA or RNA. For example,an open circle may be designed to contain several repeats of a knownarbitrary sequence, referred to as detection tags. A secondaryhybridization step can be used to bind detection probes to thesedetection tags (FIG. 7). The detection probes may be labeled asdescribed above with, for example, an enzyme, fluorescent moieties, orradioactive isotopes. By using three detection tags per open circleprobe, and four fluorescent moieties per each detection probe, one mayobtain a total of twelve fluorescent signals for every open circle proberepeat in the TS-DNA, yielding a total of 12,000 fluorescent moietiesfor every ligated open circle probe that is amplified by RCA.

(c) Multiplexing and Hybridization Array Detection

RCA is easily multiplexed by using sets of different open circle probes,each set carrying different target probe sequences designed for bindingto unique targets. Note that although the target probe sequencesdesigned for each target are different, the primer complement portionmay remain unchanged, and thus the primer for rolling circle replicationcan remain the same for all targets. Only those open circle probes thatare able to find their targets will give rise to TS-DNA. The TS-DNAmolecules generated by RCA are of high molecular weight and lowcomplexity; the complexity being the length of the open circle probe.There are two alternatives for capturing a given TS-DNA to a fixedposition in a solid-state detector. One is to include within the spacerregion of the open circle probes a unique address tag sequence for eachunique open circle probe. TS-DNA generated from a given open circleprobe will then contain sequences corresponding to a specific addresstag sequence. A second and preferred alternative is to use the targetsequence present on the TS-DNA as the address tag.

(d) Detecting Groups of Target Sequences

Multiplex RCA assays are particularly useful for detecting mutations ingenes where numerous distinct mutations are associated with certaindiseases or where mutations in multiple genes are involved. For example,although the gene responsible for Huntington's chorea has beenidentified, a wide range of mutations in different parts of the geneoccur among affected individuals. The result is that no single test hasbeen devised to detect whether an individual has one or more of the manyHuntington's mutations. A single LM-RCA assay can be used to detect thepresence of one or more members of a group of any number of targetsequences. This can be accomplished, for example, by designing an opencircle probe (and associated gap oligonucleotides, if desired) for eachtarget sequence in the group, where the target probe portions of eachopen circle probe are different but the sequence of the primer portionsand the sequence of the detection tag portions of all the open circleprobes are the same. All of the open circle probes are placed in thesame OCP-target sample mixture, and the same primer and detection probeare used to amplify and detect TS-DNA. If any of the target sequencesare present in the target sample, the OCP for that target will beligated into a circle and the circle will be amplified to form TS-DNA.Since the detection tags on TS-DNA resulting from amplification of anyof the OCPs are the same, TS-DNA resulting from ligation of any of theOCPs will be detected in that assay. Detection indicates that at leastone member of the target sequence group is present in the target sample.This allows detection of a trait associated with multiple targetsequences in a single tube or well.

If a positive result is found, the specific target sequence involved canbe identified by using a multiplex assay. This can be facilitated byincluding an additional, different detection tag in each of the OCPs ofthe group. In this way, TS-DNA generated from each different OCP,representing each different target sequence, can be individuallydetected. It is convenient that such multiple assays need be performedonly when an initial positive result is found.

The above scheme can also be used with arbitrarily chosen groups oftarget sequences in order to screen for a large number of targetsequences without having to perform an equally large number of assays.Initial assays can be performed as described above, each using adifferent group of OCPs designed to hybridize to a different group oftarget sequences. Additional assays to determine which target sequenceis present can then be performed on only those groups that produceTS-DNA. Such group assays can be further nested if desired.

(e) In Situ Detection Using RCA

In situ hybridization, and its most powerful implementation, known asfluorescent in situ hybridization (FISH), is of fundamental importancein cytogenetics. RCA can be adapted for use in FISH, as follows.

Open circle probes are ligated on targets on microscope slides, andincubated in situ with fluorescent precursors during rolling circlereplication. The rolling circle DNA polymerase displaces the ligatedopen circle probe from the position where it was originally hybridized.However, the circle will remain topologically trapped on the chromosomeunless the DNA is nicked (Nilsson et al. (1994)). The presence ofresidual chromatin may slow diffusion of the circle along thechromosome. Alternatively, fixation methods may be modified to minimizethis diffusional effect. This diffusion has an equal probability ofoccurring in either of two directions along the chromosome, and hencenet diffusional displacement may be relatively small during a 10 minuteincubation. During this time rolling circle replication should generatea linear molecule of approximately 25,000 nucleotides containingapproximately 2,500 bromodeoxyuridine moieties, which can be detectedwith a biotinylated anti-BUDR IgG (Zymed Labs, Inc.) andfluorescein-labeled avidin. This level of incorporation shouldfacilitate recording of the image using a microscope-based CCD system.Diffusion may also be limited because the TS-DNA should be able tohybridize with the complement of the target strand.

Multiplexed in situ detection can be carried out as follows: Rollingcircle replication is carried out using unlabeled nucleotides. Thedifferent TS-DNAs are then detected using standard multi-color FISH withdetection probes specific for each unique target sequence or each uniquedetection tag in the TS-DNA.

(f) Enzyme-linked Detection

Amplified nucleic acid labeled by incorporation of labeled nucleotidescan be detected with established enzyme-linked detection systems. Forexample, amplified nucleic acid labeled by incorporation ofbiotin-16-UTP (Boehringher Mannheim) can be detected as follows. Thenucleic acid is immobilized on a solid glass surface by hybridizationwith a complementary DNA oligonucleotide (address probe) complementaryto the target sequence (or its complement) present in the amplifiednucleic acid. After hybridization, the glass slide is washed andcontacted with alkaline phosphatase-streptavidin conjugate (Tropix,Inc., Bedford, Mass.). This enzyme-streptavidin conjugate binds to thebiotin moieties on the amplified nucleic acid. The slide is again washedto remove excess enzyme conjugate and the chemiluminescent substrateCSPD (Tropix, Inc.) is added and covered with a glass cover slip. Theslide can then be imaged in a Biorad Fluorimager.

2. Nested LM-RCA

After RCA, a round of LM-RCA can be performed on the TS-DNA produced inthe first RCA. This new round of LM-RCA is performed with a new opencircle probe, referred to as a secondary open circle probe, havingtarget probe portions complementary to a target sequence in the TS-DNAproduced in the first round. When such new rounds of LM-RCA areperformed, the amplification is referred to herein as nested LM-RCA.Nested LM-RCA is particularly useful for in situ hybridizationapplications of LM-RCA. Preferably, the target probe portions of thesecondary OCP are complementary to a secondary target sequence in thespacer sequences of the TS-DNA produced in the first RCA. The complementof this secondary target sequence is present in the spacer portion ofthe OCP or ATC used in the first RCA. After mixing the secondary OCPwith the TS-DNA, ligation and rolling circle amplification proceed as inLM-RCA. Each ligated secondary OCP generates a new TS-DNA. By having,for example, two secondary target sequence portions in the first roundOCP, the new round of LM-RCA will yield two secondary TS-DNA moleculesfor every OCP or ATC repeat in the TS-DNA produced in the first RCA.Thus, the amplification yield of nested LM-RCA is about 2000-fold. Theoverall amplification using two cycles of RCA is thus1000×2000=2,000,000. Nested LM-RCA can follow any DNA replication ortranscription operation described herein, such as RCA, LM-RCA, secondaryDNA strand displacement, strand displacement cascade amplification, ortranscription.

Generally, nested LM-RCA involves, following a first RCA,

(a) mixing a secondary open circle probe with the polymerase mixture,resulting in an OCP-TS mixture, and incubating the OCP-TS mixture underconditions promoting hybridization between the secondary open circleprobe and the tandem sequence DNA,

(b) mixing ligase with the OCP-TS mixture, resulting in a secondaryligation mixture, and incubating the secondary ligation mixture underconditions promoting ligation of the secondary open circle probe to forma secondary amplification target circle,

(c) mixing a rolling circle replication primer with the secondaryligation mixture, resulting in a secondary primer-ATC mixture, andincubating the secondary primer-ATC mixture under conditions thatpromote hybridization between the secondary amplification target circleand rolling circle replication primer,

(d) mixing DNA polymerase with the secondary primer-ATC mixture,resulting in a secondary polymerase-ATC mixture, and incubating thesecondary polymerase-ATC mixture under conditions promoting replicationof the secondary amplification target circle, where replication of thesecondary amplification target circle results in formation of nestedtandem sequence DNA.

An exonuclease digestion step can be added prior to performing thenested LM-RCA. This is especially useful when the target probe portionsof the secondary open circle probe are the same as those in the firstopen circle probe. Any OCP which has been ligated will not be digestedsince ligated OCPs have no free end. A preferred way to digest OCPs thathave hybridized to TS-DNA during the first round of LM-RCA is to use aspecial rolling circle replication primer containing at least about fourphosphorothioate linkages between the nucleotides at the 5′ end. Then,following rolling circle replication, the reaction mixture is subjectedto exonuclease digestion. By using a 5′ exonuclease unable to cleavethese phosphorothioate linkages, only the OCPs hybridized to TS-DNA willbe digested, not the TS-DNA. The TS-DNA generated during the first cycleof amplification will not be digested by the exonuclease because it isprotected by the phosphorothioate linkages at the 5′ end. A preferredexonuclease for this purpose is the T7 gene 6 exonuclease. The T7 gene 6exonuclease can be inactivated prior to adding the secondary open circleprobe by heating to 90° C. for 10 minutes.

By using an exonuclease digestion, nested LM-RCA can be performed usingthe same target sequence used in a first round of LM-RCA. This can bedone, for example, generally as follows. After the first round ofLM-RCA, the unligated open circle probes and gap oligonucleotideshybridized to TS-DNA are digested with T7 gene 6 exonuclease. Theexonuclease is inactivated by heating for 10 minutes at 90° C. Then asecond open circle probe is added. In this scheme, the second opencircle probe has target probe portions complementary to the sameoriginal target sequence, but which contain a different (arbitrary)spacer region sequence. A second round of LM-RCA is then performed. Inthis second round, the target of the second open circle probes comprisesthe repeated target sequences contained in the TS-DNA generated by thefirst cycle. This procedure has the advantage of preserving the originaltarget sequence in the amplified DNA obtained after nested LM-RCA.

Nested LM-RCA can also be performed on ligated OCPs or ATCs that havenot been amplified. In this case, LM-RCA can be carried out using eitherATCs or target-dependent ligated OCPs. This is especially useful for insitu detection. For in situ detection, the first, unamplified OCP, whichis topologically locked to its target sequence, can be subjected tonested LM-RCA. By not amplifying the first OCP, it can remain hybridizedto the target sequence while LM-RCA amplifies a secondary OCPtopologically locked to the first OCP. This is illustrated in FIG. 12.

3. Secondary DNA Strand Displacement and Strand Displacement CascadeAmplification

Secondary DNA strand displacement is another way to amplify TS-DNA.Secondary DNA strand displacement is accomplished by hybridizingsecondary DNA strand displacement primers to TS-DNA and allowing a DNApolymerase to synthesize DNA from these primed sites (FIG. 11). Since acomplement of the secondary DNA strand displacement primer occurs ineach repeat of the TS-DNA, secondary DNA strand displacement can resultin a level of amplification similar to or larger than that obtained inRCT. The product of secondary DNA strand displacement is referred to assecondary tandem sequence DNA or TS-DNA-2.

Secondary DNA strand displacement can be accomplished by performing RCAto produce TS-DNA in a polymerase-ATC mixture, and then mixing secondaryDNA strand displacement primer with the polymerase-ATC mixture,resulting in a secondary DNA strand displacement mixture, and incubatingthe secondary DNA strand displacement mixture under conditions promotingreplication of the tandem sequence DNA. The secondary DNA stranddisplacement primer is complementary to a part of the OCP or ATC used togenerated TS-DNA as described earlier. It is preferred that thesecondary DNA strand displacement primer is not complementary to therolling circle replication primer, or to a tertiary DNA stranddisplacement primer, if used.

Secondary DNA strand displacement can also be carried out simultaneouslywith rolling circle replication. This is accomplished by mixingsecondary DNA strand displacement primer with the polymerase-ATC mixtureprior to incubating the mixture for rolling circle replication. Forsimultaneous rolling circle replication and secondary DNA stranddisplacement, it is preferred that the rolling circle DNA polymerase beused for both replications. This allows optimum conditions to be usedand results in displacement of other strands being synthesizeddownstream as shown in FIG. 11B. Secondary DNA strand displacement canfollow any DNA replication operation disclosed herein, such as RCA,LM-RCA or nested LM-RCA.

To optimize the efficiency of secondary DNA strand displacement, it ispreferred that a sufficient concentration of secondary DNA stranddisplacement primer be used to obtain sufficiently rapid priming of thegrowing TS-DNA strand to outcompete any remaining unligated OCPs and gapoligonucleotides that might be present for binding to TS-DNA. Ingeneral, this is accomplished when the secondary DNA strand displacementprimer is in very large excess compared to the concentration ofsingle-stranded sites for hybridization of the secondary DNA stranddisplacement primer on TS-DNA. Optimization of the concentration ofsecondary DNA strand displacement primer can be aided by analysis ofhybridization kinetics using methods such as those described by Youngand Anderson, “Quantitative analysis of solution hybridization” inNucleic Acid Hybridization: A Practical Approach (IRL Press, 1985) pages47-71. For example, assuming that φ29 DNA polymerase is used as therolling circle DNA polymerase, TS-DNA is generated at a rate of about 53nucleotides per second, and the rolling circle DNA polymerase generatesapproximately 10 copies of the amplification target circle. Analysis ofthe theoretical solution hybridization kinetics for an OCP driver DNA(unligated OCP) present at a concentration of 80 nM (a typicalconcentration used for a LM-RCA ligation operation), and the theoreticalsolution hybridization kinetics for a secondary DNA strand displacementprimer driver DNA present at a concentration of 800 nM, indicates thatthe secondary DNA strand displacement primer will bind to those 10copies within 30 seconds, while unligated OCP will hybridize to lessthan one site in 30 seconds (8% of sites filled). If the concentrationof DNA polymerase is relatively high (for this example, in the range of100 to 1000 nM), the polymerase will initiate DNA synthesis at eachavailable 3′ terminus on the hybridized secondary DNA stranddisplacement primers, and these elongating TS-DNA-2 molecules will blockany hybridization by the unligated OCP molecules. Alternatively, theefficiency of secondary DNA strand displacement can be improved by theremoval of unligated open circle probes and gap oligonucleotides priorto amplification of the TS-DNA. In secondary DNA strand displacement, itis preferred that the concentration of secondary DNA strand displacementprimer generally be from 500 nM to 5000 nM, and most preferably from 700nM to 1000 nM.

As a secondary DNA strand displacement primer is elongated, the DNApolymerase will run into the 5′ end of the next hybridized secondary DNAstrand displacement molecule and will displace its 5′ end. In thisfashion a tandem queue of elongating DNA polymerases is formed on theTS-DNA template. As long as the rolling circle reaction continues, newsecondary DNA strand displacement primers and new DNA polymerases areadded to TS-DNA at the growing end of the rolling circle. The generationof TS-DNA-2 and its release into solution by strand displacement isshown diagrammatically in FIG. 11.

Generally, secondary DNA strand displacement can be performed by,simultaneous with or following RCA, mixing a secondary DNA stranddisplacement primer with the polymerase-ATC mixture, and incubating thepolymerase-ATC mixture under conditions that promote both hybridizationbetween the tandem sequence DNA and the secondary DNA stranddisplacement primer, and replication of the tandem sequence DNA, wherereplication of the tandem sequence DNA results in the formation ofsecondary tandem sequence DNA.

When secondary DNA strand displacement is carried out in the presence ofa tertiary DNA strand displacement primer, an exponential amplificationof TS-DNA sequences takes place. This special and preferred mode ofsecondary DNA strand displacement is referred to as strand displacementcascade amplification (SDCA). In SDCA, illustrated in FIG. 13, asecondary DNA strand displacement primer primes replication of TS-DNA toform TS-DNA-2, as described above. The tertiary DNA strand displacementprimer strand can then hybridize to, and prime replication of, TS-DNA-2to form TS-DNA-3. Strand displacement of TS-DNA-3 by the adjacent,growing TS-DNA-3 strands makes TS-DNA-3 available for hybridization withsecondary DNA strand displacement primer. This results in another roundof replication resulting in TS-DNA-4 (which is equivalent to TS-DNA-2).TS-DNA-4, in turn, becomes a template for DNA replication primed bytertiary DNA strand displacement primer. The cascade continues thismanner until the reaction stops or reagents become limiting. Thisreaction amplifies DNA at an almost exponential rate, although kineticsare not truly exponential because there are stochastically distributedpriming failures, as well as steric hindrance events related to thelarge size of the DNA network produced during the reaction. In apreferred mode of SDCA, the rolling circle replication primer serves asthe tertiary DNA strand displacement primer, thus eliminating the needfor a separate primer. For this mode, the rolling circle replicationprimer should be used at a concentration sufficiently high to obtainrapid priming on the growing TS-DNA-2 strands. To optimize theefficiency of SDCA, it is preferred that a sufficient concentration ofsecondary DNA strand displacement primer and tertiary DNA stranddisplacement primer be used to obtain sufficiently rapid priming of thegrowing TS-DNA strand to outcompete TS-DNA for binding to itscomplementary TS-DNA, and, in the case of secondary DNA stranddisplacement primer, to outcompete any remaining unligated OCPs and gapoligonucleotides that might be present for binding to TS-DNA. Ingeneral, this is accomplished when the secondary DNA strand displacementprimer and tertiary DNA strand displacement primer are both in verylarge excess compared to the concentration of single-stranded sites forhybridization of the DNA strand displacement primers on TS-DNA. Forexample, it is preferred that the secondary DNA strand displacementprimer is in excess compared to the concentration of single-strandedsecondary DNA strand displacement primer complement sites on TS-DNA,TS-DNA-3, TS-DNA-5, and so on. In the case of tertiary DNA stranddisplacement primer, it is preferred that the tertiary DNA stranddisplacement primer is in excess compared to the concentration ofsingle-stranded tertiary DNA strand displacement primer complement siteson TS-DNA-2, TS-DNA-4, TS-DNA-6, and so on. Such an excess generallyresults in a primer hybridizing to its complement in TS-DNA beforeamplified complementary TS-DNA can hybridize. Optimization of primerconcentrations can be aided by analysis of hybridization kinetics (Youngand Anderson). In a strand displacement cascade amplification, it ispreferred that the concentration of both secondary and tertiary DNAstrand displacement primers generally be from 500 nM to 5000 nM, andmost preferably from 700 nM to 1000 nM.

As in the case of secondary DNA strand displacement primers, if theconcentration of DNA polymerase is sufficiently high, the polymerasewill initiate DNA synthesis at each available 3′ terminus on thehybridized tertiary DNA strand displacement primers, and theseelongating TS-DNA-3 molecules will block any hybridization by TS-DNA-2.As a tertiary DNA strand displacement primer is elongated to formTS-DNA-3, the DNA polymerase will run into the 5′ end of the nexthybridized tertiary DNA strand displacement primer molecule and willdisplace its 5′ end. In this fashion a tandem queue of elongating DNApolymerases is formed on the TS-DNA-2 template. As long as the reactioncontinues, new rolling circle replication primers and new DNApolymerases are added to TS-DNA-2 at the growing ends of TS-DNA-2. Thishybridization/replication/strand displacement cycle is repeated withhybridization of secondary DNA strand displacement primers on thegrowing TS-DNA-3. The cascade of TS-DNA generation, and their releaseinto solution by strand displacement is shown diagrammatically in FIG.13.

Generally, strand displacement cascade amplification can be performedby, simultaneous with, or following, RCA, mixing a secondary DNA stranddisplacement primer and a tertiary DNA strand displacement primer withthe polymerase-ATC mixture, and incubating the polymerase-ATC mixtureunder conditions that promote both hybridization between the tandemsequence DNA and the DNA strand displacement primers, and replication ofthe tandem sequence DNA, where replication of the tandem sequence DNAresults in the formation of secondary tandem sequence DNA.

An example of the amplification yield generated by a strand displacementcascade amplification can be roughly estimated as follows. A rollingcircle reaction that proceeds for 35 minutes at 53 nucleotides persecond will generate 1236 copies of a 90 nucleotide amplification targetcircle. Thus, TS-DNA-1 contains 1236 tandem repeats. As these 1236tandem repeats grow, priming and synthesis with secondary DNA stranddisplacement primers can generate at least 800 TS-DNA-2 molecules,taking into account delays and missed priming events. These newmolecules will have lengths linearly distributed in the range of 1 to799 repeats. Next, priming events on TS-DNA-2 by tertiary DNA stranddisplacement primers can generate at least 500 TS-DNA-3 molecules,taking into account delays and missed priming events, and these newmolecules will have lengths linearly distributed in the range of 1 to499 repeats. Then, priming events on TS-DNA-3 by secondary DNA stranddisplacement primers can generate at least 300 TS-DNA-4 molecules,taking into account delays and missed priming events, and these newmolecules will have lengths linearly distributed in the range of 1 to299 repeats. A conservative overall amplification yield, calculated asthe product of only these four amplification levels, is estimated to be1.86×10¹⁰ repeats of the original OCP or ATC. Thus, SDCA is capable ofextremely high amplification yields in an isothermal 35-minute reaction.

A modified form of secondary DNA strand displacement results inamplification of TS-DNA and is referred to as opposite strandamplification (OSA). OSA is the same as secondary DNA stranddisplacement except that a special form of rolling circle replicationprimer is used that prevents it from hybridizing to TS-DNA-2. This canbe accomplished in a number of ways. For example, the rolling circlereplication primer can have an affinity tag coupled to itsnon-complementary portion allowing the rolling circle replication primerto be removed prior to secondary DNA strand displacement. Alternatively,remaining rolling circle replication primer can be crippled followinginitiation of rolling circle replication. One preferred form of rollingcircle replication primer for use in OSA is designed to form a hairpinthat contains a stem of perfectly base-paired nucleotides. The stem cancontain 5 to 12 base pairs, most preferably 6 to 9 base pairs. Such ahairpin-forming rolling circle replication primer is a poor primer atlower temperature (less than 40° C.) because the hairpin structureprevents it from hybridizing to complementary sequences. The stem shouldinvolve a sufficient number of nucleotides in the complementary portionof the rolling circle replication primer to interfere with hybridizationof the primer to the OCP or ATC. Generally, it is preferred that a steminvolve 5 to 24 nucleotides, and most preferably 6 to 18 nucleotides, ofthe complementary portion of a rolling circle replication primer. Arolling circle replication primer where half of the stem involvesnucleotides in the complementary portion of the rolling circlereplication primer and the other half of the stem involves nucleotidesin the non-complementary portion of the rolling circle replicationprimer is most preferred. Such an arrangement eliminates the need forself-complementary regions in the OCP or ATC when using ahairpin-forming rolling circle replication primer.

When starting the rolling circle replication reaction, secondary DNAstrand displacement primer and rolling circle replication primer areadded to the reaction mixture, and the solution is incubated briefly ata temperature sufficient to disrupt the hairpin structure of the rollingcircle replication primer but to still allow hybridization to the primercomplement portion of the amplification target circle (typically greaterthan 50° C.). This incubation permits the rolling circle replicationprimer to hybridize to the primer complement portion of theamplification target circle. The solution is then brought to the propertemperature for rolling circle replication, and the rolling circle DNApolymerase is added. As the rolling circle reaction proceeds, TS-DNA isgenerated, and as the TS-DNA grows in length, the secondary DNA stranddisplacement primer rapidly initiates DNA synthesis with multiple stranddisplacement reactions on TS-DNA. These reactions generate TS-DNA-2,which is complementary to the TS-DNA. While TS-DNA-2 contains sequencescomplementary to the rolling circle replication primer, the primer isnot able to hybridize nor prime efficiently at the reaction temperaturedue to its hairpin structure at this temperature. Thus, there is nofurther priming by the rolling circle replication primer and the onlyproducts generated are TS-DNA and TS-DNA-2. The reaction comes to a haltas rolling circle amplification stops and TS-DNA becomes completelydouble-stranded. In the course of the reaction, an excess ofsingle-stranded TS-DNA-2 is generated.

Another form of rolling circle replication primer useful in OSA is achimera of DNA and RNA. In this embodiment, the rolling circle primerhas deoxyribonucleotides at its 3′ end and ribonucleotides in theremainder of the primer. It is preferred that the rolling circlereplication primer have five or six deoxyribonucleotides at its 3′ end.By making part of the rolling circle replication primer withribonucleotide, the primer can be selectively degraded by RNAse H whenit is hybridized to DNA. Such hybrids form during OSA as TS-DNA-2 issynthesized. The deoxyribonucleotides at the 3′ end allow the rollingcircle DNA polymerase to initiate rolling circle replication. RNAse Hcan then be added to the OSA reaction to prevent priming of TS-DNA-2replication.

An example of the amplification yield generated by OSA can be roughlyestimated as follows. A rolling circle reaction that proceeds for 45minutes at 53 nucleotides per second will generate tandem 1590 copies ofa 90 nucleotide amplification target circle. Thus, TS-DNA-1 contains1590 tandem repeats. As these 1590 tandem repeats grow, priming anddisplacement reactions with secondary DNA strand displacement primerswill generate and release up to 1400 TS-DNA-2 molecules, and those newmolecules will have lengths linearly distributed in the range of 1 to1399 repeats. Calculations indicate that after 45 minutes,single-stranded TS-DNA-2 exceeds the amount of TS-DNA by a factor ofabout 700. OSA is useful for generating single-stranded DNA thatcontains the reverse complement of the target sequence. Overallamplification can be of the order of one million fold.

If secondary DNA strand displacement is used with a ligated OCP,unligated OCPs and gap oligonucleotides may be removed prior to rollingcircle replication to eliminate competition between unligated OCPs andgap oligonucleotides and the secondary DNA strand displacement primerfor hybridization to TS-DNA. An exception would be when secondary DNAstrand displacement is used in conjunction with gap-filling LM-RCA, asdescribed below. Alternatively, the concentration of the secondary DNAstrand displacement primer can be made sufficiently high so that itoutcompetes unligated OCP for hybridization to TS-DNA. This allowssecondary DNA strand displacement to be performed without removal ofunligated OCPs.

The DNA generated by secondary DNA strand displacement can be labeledand/or detected using the same labels, labeling methods, and detectionmethods described for use with TS-DNA. Most of these labels and methodsare adaptable for use with nucleic acids in general. A preferred methodof labeling the DNA is by incorporation of labeled nucleotides duringsynthesis.

4. Multiple Ligation Cycles

Using a thermostable DNA ligase, such as AMPLIGASE® (EpicentreTechnologies, Inc.), the open circle probe ligation reaction may becycled a number of times between a annealing temperature (55° C.) and amelting temperature (96° C.). This cycling will produce multipleligations for every target sequence present in the sample. For example,8 cycles of ligation would provide and approximate 6-fold increase inthe number of ligated circles. A preferred cycling protocol is 96° C.for 2 seconds, 55° C. for 2 seconds, and 60° C. for 70 seconds in aPerkin Elmer 9600 thermal cycler. If the number of cycles is kept small,the linearity of the amplification response should not be compromised.

The expected net amplification yield using eight ligation cycles,secondary fluorescent tags, and array hybridization can be calculated asshown below.

Ligation cycling yield: 6 OSA yield 1,000,000 number of fluorescenttags/circle 5 20% array hybridization yield 0.2Net yield=6×1,000,000×5×0.2=6,000,000target molecules×6,000,000=6×10⁸ fluors bound on the surface

5. Transcription Following RCA (RCT)

Once TS-DNA is generated using RCA, further amplification can beaccomplished by transcribing the TS-DNA from promoters embedded in theTS-DNA. This combined process, referred to as rolling circle replicationwith transcription (RCT), or ligation mediated rolling circlereplication with transcription (LM-RCT), requires that the OCP or ATCfrom which the TS-DNA is made have a promoter portion in its spacerregion. The promoter portion is then amplified along with the rest ofthe OCP or ATC resulting in a promoter embedded in each tandem repeat ofthe TS-DNA (FIG. 8). Since transcription, like rolling circleamplification, is a process that can go on continuously (withre-initiation), multiple transcripts can be produced from each of themultiple promoters present in the TS-DNA. RCT effectively adds anotherlevel of amplification of ligated OCP sequences.

Generally, RCT can be accomplished by performing RCA to produce TS-DNAin a polymerase-OCP mixture or polymerase-ATC mixture, and then mixingRNA polymerase with the polymerase-OCP mixture or polymerase-ATCmixture, resulting in a transcription mixture, and incubating thetranscription mixture under conditions promoting transcription of thetandem sequence DNA. The OCP or ATC must include the sequence of apromoter for the RNA polymerase (a promoter portion) in its spacerregion for RCT to work. The transcription step in RCT generally can beperformed using established conditions for in vitro transcription of theparticular RNA polymerase used. Preferred conditions are described inthe Examples. Alternatively, transcription can be carried outsimultaneously with rolling circle replication. This is accomplished bymixing RNA polymerase with the polymerase-OCP mixture or polymerase-ATCmixture prior to incubating the mixture for rolling circle replication.For simultaneous rolling circle replication and transcription therolling circle DNA polymerase and RNA polymerase must be active in thesame conditions. Such conditions can be optimized in order to balanceand/or maximize the activity of both polymerases. It is not necessarythat the polymerase achieve their maximum activity, a balance betweenthe activities is preferred. Transcription can follow any DNAreplication operation described herein, such as RCA, LM-RCA, nestedLM-RCA, secondary DNA strand displacement, or strand displacementcascade amplification.

The transcripts generated in RCT can be labeled and/or detected usingthe same labels, labeling methods, and detection methods described foruse with TS-DNA. Most of these labels and methods are adaptable for usewith nucleic acids in general. A preferred method of labeling RCTtranscripts is by direct labeling of the transcripts by incorporation oflabeled nucleotides, most preferably biotinylated nucleotides, duringtranscription.

6. Gap-Filling Ligation

The gap space formed by an OCP hybridized to a target sequence isnormally occupied by one or more gap oligonucleotides as describedabove. Such a gap space may also be filled in by a gap-filling DNApolymerase during the ligation operation. This modified ligationoperation is referred to herein as gap-filling ligation and is thepreferred form of the ligation operation. The principles and procedurefor gap-filling ligation are generally analogous to the filling andligation performed in gap LCR (Wiedmann et al., PCR Methods andApplications (Cold Spring Harbor Laboratory Press, Cold Spring HarborLaboratory, NY, 1994) pages S51-S64; Abravaya et al., Nucleic AcidsRes., 23(4):675-682 (1995); European Patent Application EP0439182(1991)). In the case of LM-RCA, the gap-filling ligation operation issubstituted for the normal ligation operation. Gap-filling ligationprovides a means for discriminating between closely related targetsequences. An example of this is described in Example 3. Gap-fillingligation can be accomplished by using a different DNA polymerase,referred to herein as a gap-filling DNA polymerase. Suitable gap-fillingDNA polymerases are described above. Alternatively, DNA polymerases ingeneral can be used to fill the gap when a stop base is used. The use ofstop bases in the gap-filling operation of LCR is described in EuropeanPatent Application EP0439182. The principles of the design of gaps andthe ends of flanking probes to be joined, as described in EP0439182, isgenerally applicable to the design of the gap spaces and the ends oftarget probe portions described herein.

To prevent interference of the gap-filling DNA polymerase with rollingcircle replication, the gap-filling DNA polymerase can be removed byextraction or inactivated with a neutralizing antibody prior toperforming rolling circle replication. Such inactivation is analogous tothe use of antibodies for blocking Taq DNA polymerase prior to PCR(Kellogg et al., Biotechniques 16(6):1134-1137 (1994)).

Gap-filling ligation is also preferred because it is highly compatiblewith exponential amplification of OCP sequences similar to the stranddisplacement cascade amplification (SDCA) as described above. As TS-DNAis formed during rolling circle replication, unligated OCP moleculespresent in the reaction hybridize to TS-DNA, leaving gap spaces betweenevery OCP repeat. The hybridized OCP molecules serve as primers forsecondary DNA synthesis.

Generally, gap-filling LM-RCA can be performed by, in an LM-RCAreaction, (1) using a target sequence with a central region locatedbetween a 5′ region and a 3′ region, and an OCP where neither the lefttarget probe portion of the open circle probe nor the right target probeportion of the open circle probe is complementary to the central regionof the target sequence, and (2) mixing gap-filling DNA polymerase withthe OCP-target sample mixture.

D. Discrimination Between Closely Related Target Sequences

Open circle probes, gap oligonucleotides, and gap spaces can be designedto discriminate closely related target sequences, such as geneticalleles. Where closely related target sequences differ at a singlenucleotide, it is preferred that open circle probes be designed with thecomplement of this nucleotide occurring at one end of the open circleprobe, or at one of the ends of the gap oligonucleotide(s). Ligation ofgap oligonucleotides with a mismatch at either terminus is extremelyunlikely because of the combined effects of hybrid instability andenzyme discrimination. When the TS-DNA is generated, it will carry acopy of the gap oligonucleotide sequence that led to a correct ligation.Gap oligonucleotides may give even greater discrimination betweenrelated target sequences in certain circumstances, such as thoseinvolving wobble base pairing of alleles. Features of open circle probesand gap oligonucleotides that increase the target-dependency of theligation operation are generally analogous to such features developedfor use with the ligation chain reaction. These features can beincorporated into open circle probes and gap oligonucleotides for use inLM-RCA. In particular, European Patent Application EP0439182 describesseveral features for enhancing target-dependency in LCR that can beadapted for use in LM-RCA. The use of stop bases in the gap space, asdescribed in European Patent Application EP0439182, is a preferred modeof enhancing the target discrimination of a gap-filling ligationoperation.

A preferred form of target sequence discrimination can be accomplishedby employing two types of open circle probes. These two OCPs would bedesigned essentially as shown in FIG. 2, with small modifications. Inone embodiment, a single gap oligonucleotide is used which is the samefor both target sequences, that is, the gap oligonucleotide iscomplementary to both target sequences. In a preferred embodiment, agap-filling ligation operation can be used (Example 3). Target sequencediscrimination would occur by virtue of mutually exclusive ligationevents, or extension-ligation events, for which only one of the twoopen-circle probes is competent. Preferably, the discriminatornucleotide would be located at the penultimate nucleotide from the 3′end of each of the open circle probes. The two open circle probes wouldalso contain two different detection tags designed to bind alternativedetection probes and/or address probes. Each of the two detection probeswould have a different detection label. Both open circle probes wouldhave the same primer complement portion. Thus, both ligated open circleprobes can be amplified using a single primer. Upon array hybridization,each detection probe would produce a unique signal, for example, twoalternative fluorescence colors, corresponding to the alternative targetsequences.

E. Optimization of RCA

1. Assay Background

A potential source of background signals is the formation of circularmolecules by non-target-directed ligation events. The contribution ofsuch events to background signals can be minimized using fivestrategies, alone or in combination, as follows:

(a) The use of a thermostable DNA ligase such as AMPLIGASE® (Kalin etal. (1992)) or the T. thermophilus DNA ligase (Barany (1991)) willminimize the frequency of non-target-directed ligation events becauseligation takes place at high temperature (50 to 75° C.).

(b) In the case of in situ hybridization, ligation of the open circleprobe to the target sequence permits extensive washing. This washingwill remove any circles that may have been formed by non-target-directedligation, while circles ligated on-target are impossible to removebecause they are topologically trapped (Nilsson et al. (1994)).

(c) The use of one or more gap oligonucleotides provides additionalspecificity in the ligation event. Using a gap oligonucleotide greatlyreduces the probability of non-target-directed ligation. Particularlyfavored is the use of a gap oligonucleotide, or a gap-filling ligationoperation, coupled to a capture hybridization step where thecomplementary portion of an address probe spans the ligation junction ina highly discriminatory fashion, as shown below and in FIG. 6.

   complement of gap oligonucleotide (11 nucleotides)        \           / ...NNNTA{GTCAGATCAGA}TANNNNN... TS-DNA      || ||||||||||| ||       AT CAGTCTAGTCT ATNNNNN... address probe     /                 \complementary portion of address probe (15 nucleotides hybridized)Brackets ({ }) mark sequence complementary to the gap oligonucleotide.The TS-DNA shown is SEQ ID NO:10 and the address probe sequence shown isSEQ ID NO:4. This system can be used with gap oligonucleotides of anylength. Where the gap between the ends of an open circle probehybridized to a target sequence is larger than the desired address probelength, an address probe can be designed to overlap just one of thejunctions between the gap sequence and the open circle probe sequence.

The capture step involves hybridization of the amplified DNA to anaddress probe via a specific sequence interaction at the ligationjunction, involving the complement of the gap oligonucleotide, as shownabove. Guo et al. (1994), have shown that 15-mer oligonucleotides boundcovalently on glass slides using suitable spacers, can be used tocapture amplified DNA with reasonably high efficiency. This system canbe adapted to detection of amplified nucleic acid (TS-DNA or TS-RNA) byusing address probes to capture the amplified nucleic acid. In theexample shown above, only LM-RCA amplified DNA generated from correctligation events will be captured on the solid-state detector.

Optionally one may use additional immobilizing reagents, known in theart as capture probes (Syvanen et al., Nucleic Acids Res., 14:5037(1986)) in order to bind nucleic acids containing the target sequence toa solid surface. Suitable capture probes contain biotinylatedoligonucleotides (Langer et al. (1981)) or terminal biotin groups.Immobilization may take place before or after the ligation reaction.Immobilization serves to allow removal of unligated open circle probesas well as non-specifically ligated circles.

(d) Using ligation conditions that favor intramolecular ligation.Conditions are easily found where circular ligation of OCPs occurs muchmore frequently than tandem linear ligation of two OCPs. For example,circular ligation is favored when the temperature at which the ligationoperation is performed is near the melting temperature (T_(m)) of theleast stable of the left target probe portion and the right target probeportion when hybridized to the target sequence. When ligation is carriedout near the T_(m) of the target probe portion with the lowest T_(m),the target probe portion is at association/dissociation equilibrium. Atequilibrium, the probability of association in cis (that is, with theother target probe portion of the same OCP) is much higher than theprobability of association in trans (that is, with a different OCP).When possible, it is preferred that the target probe portions bedesigned with melting temperatures near suitable temperatures for theligation operation. The use of a thermostable ligase, however, allows awide range of ligation temperatures to be used, allowing greater freedomin the selection of target sequences.

(e) Peptide nucleic acids form extremely stable hybrids with DNA, andhave been used as specific blockers of PCR reactions (Orum et al.,Nucleic Acids Res., 21:5332-5336 (1993)). A special PNA probe, referredto herein as a PNA clamp, can be used to block rolling circleamplification of OCPs that have been ligated illegitimately (that is,ligated in the absence of target). By using one or more gapoligonucleotides during ligation, or by using gap-filling ligation,illegitimately ligated circles will lack the gap sequence and they canbe blocked with a PNA clamp that is complementary to the sequenceresulting from the illegitimate ligation of the 3′ end and the 5′ end ofthe OCP. This is illustrated in the diagram below, where the PNA clampllllrrrr is positioned exactly over the junction:

         llllrrrr PNA clamp          |||||||| ...LLLLLLLLLLRRRRRRRRRR...ligated OCP             /\      ligation siteIn this diagram, “L” and “l” represent a nucleotide in the left targetprobe portion of the OCP and its complement in the PNA clamp, and “R”and “r” represent a nucleotide in the right target probe portion of theOCP and its complement in the PNA clamp. The most preferred length for aPNA clamp is 8 to 10 nucleotides. The PNA clamp is incapable ofhybridizing to unligated OCP because it can only form four to five basepairs with either target probe portion, and it is also incapable ofhybridizing with correctly ligated OCP because a gap sequence ispresent. However, the PNA clamp will hybridize strongly withillegitimately ligated OCP, and it will block the progress of therolling circle reaction because the DNA polymerase is incapable ofdisplacing a hybridized PNA molecule. This prevents amplification ofillegitimately ligated OCPs.

2. Removing Excess Unligated OCPs

The gene 6 exonuclease of phage T7 provides a useful tool for theelimination of excess open circle probes and excess gap oligonucleotidesthat will bind to the TS-DNA or LM-RCT transcripts and interfere withits hybridization to detection probes. This exonuclease digests DNAstarting from the 5′-end of a double-stranded structure. It has beenused successfully for the generation of single-stranded DNA after PCRamplification (Holloway et al., Nucleic Acids Res. 21:3905-3906 (1993);Nikiforov et al., PCR Methods and Applications 3:285-291(1994)). In anLM-RCA assay this enzyme can be added after ligation, together with therolling circle DNA polymerase. To protect TS-DNA from degradation, therolling circle replication primer can contain 3 or 4 phosphorothioatelinkages at the 5′ end, to make this molecule resistant to theexonuclease (Nikiforov et al. (1994)). The exonuclease will degradeexcess open circle probe molecules as they become associated with therolling circle DNA product. The use of this nuclease eliminates the needfor capture probes as well as the need for washing to remove excessprobes. In general, such a nuclease digestion should not be used whenperforming LM-RCT, since unligated OCPs and gap oligonucleotides areneeded to form a double-stranded transcription template with the TS-DNA.This nuclease digestion is a preferred method of eliminating unligatedOCPs and gap oligonucleotides when nested LM-RCA is to be performed.

EXAMPLES Example 1 Target-mediated Ligation of Open Circle Probes andRolling Circle Replication of Ligated Open Circle Probes

1. Ligation of Open Circle Probes

Linear oligonucleotides with 5′-phosphates are efficiently ligated byligase in the presence of a complementary target sequence. Inparticular, open circle probes hybridized to a target sequence as shownin FIG. 1, and open circle probes with gap oligonucleotides hybridizedto a target sequence as shown in as shown in FIG. 2, are readilyligated. The efficiency of such ligation can be measured by LM-RCA.

The following is an example of target-dependent ligation of an opencircle probe:

A DNA sample (target sample) is heat-denatured for 3 minutes at 95° C.,and incubated under ligation conditions (45 minutes at 60° C.) in abuffer consisting of 20 mM Tris-HCl (pH 8.2), 25 mM KCl, 10 mM MgCl₂,0.5 mM NAD, 0.05% Triton X-100, in the presence of (a) DNA ligase(AMPLIGASE®, Epicentre Technologies) at a concentration of 1 unit per 50μl, and (b) the following 5′-phosphorylated oligonucleotides:

     Open circle probe (111 nucleotides):5′-GCCTGTCCAGGGATCTGCTCAAGACTCGTCATGTCTCAGTAGCTTCTAACGGTCACAAGCTTCTAACGGTCACAAGCTTCTAACGGTCACATGTCTGCTGCCCT CTGTATT-3′ (SEQ IDNO: 1)      Gap oligonucleotide: 5′-CCTT-3′

This results in hybridization of the open circle probe and gapoligonucleotide to the target sequence, if present in the target sample,and ligation of the hybridized open circle probe and gapoligonucleotide.

2. Measuring the Rate of Rolling Circle Replication

(a) On large template: 7 kb single-stranded phage M13 circle

The rate of oligonucleotide-primed rolling circle replication onsingle-stranded M13 circles mediated by any DNA polymerase can bemeasured by using the assay described by Blanco et al., J. Biol. Chem.264:8935-8940 (1989). The efficiency of primed synthesis by the φ29 DNApolymerase is stimulated about 3-fold in the presence of Gene-32protein, a single-stranded DNA binding protein.

(b) On small templates: 110-nucleotide ligated open circle probes

The rate of oligonucleotide-primed rolling circle replication onsingle-stranded small circles of 110 bases was measured using the φ29DNA polymerase generally as described in Example 2. After five minutesof incubation, the size of the DNA product is approximately 16kilobases. This size corresponds to a polymerization rate of 53nucleotides per second. The rate of synthesis with other DNA polymerasescan be measured and optimized using a similar assay, as described byFire and Xu, Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995). It ispreferred that single-stranded circles of 110 nucleotides be substitutedfor the 34 nucleotide circles of Fire and Xu.

The φ29 DNA polymerase provides a rapid rate of polymerization of theφ29 rolling circle reaction on 110 nucleotide circular templates. At theobserved rate of 50 nucleotides per second, a 35 minute polymerizationreaction will produce a DNA product of approximately 105,000 bases. Thiswould yield an amplification of 954-fold over the original 110-basetemplate. Fire and Xu (1995) shows that rolling circle reactionscatalyzed by bacterial DNA polymerases may take place on very smallcircular templates of only 34 nucleotides. On the basis of the resultsof Fire and Yu, rolling circle replication can be carried out usingcircles of less than 90 nucleotides.

Example 2 Detection of a Mutant Ornithine Transcarbamylase (OTC) GeneUsing LM-RCA Followed by Transcription (LM-RCT)

This example describes detection of human DNA containing a mutant form(G to C) at position 114 of exon 9 of the ornithine transcarbamylasegene (Hata et al., J. Biochem. 103:302-308 (1988)). Human DNA for theassay is prepared by extraction from buffy coat using a standard phenolprocedure.

-   1. Two DNA samples (400 ng each) are heat-denatured for 4 minutes at    97° C., and incubated under ligation conditions in the presence of    two 5′-phosphorylated oligonucleotides, an open circle probe and one    gap oligonucleotide:

Open circle probe (95 nucleotides):

5′-GAGGAGAATAAAAGTTTCTCATAAGACTCGTCATGTCTCAGCAGCTTCTA (SEQ ID NO: 5)ACGGTCACTAATACGACTCACTATAGGTTCTGCCTCTGGGAACAC-3′

Gap oligonucleotide for mutant gene (8 nucleotides)

5′-TAGTGATG-3′

Gap oligonucleotide for wild type gene (8 nucleotides)

5′-TAGTGATC-3′T4 DNA ligase (New England Biolabs) is present at a concentration of 5units per μl, in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.20 MNaCl, 10 mM MgCl₂, 2 mM ATP. The concentration of open circle probe is80 nM, and the concentration of gap oligonucleotide is 100 nM. The totalvolume is 40 μl. Ligation is carried out for 25 minutes at 37° C.

-   2. 25 μl are taken from each of the above reactions and mixed with    an equal volume of a buffer consisting of 50 mM Tris-HCl (pH 7.5),    10 mM MgCl₂, 1 mM DTT, 400 μM each of dTTP, dATP, dGTP, dCTP, which    contains an 18-base rolling circle replication primer    5′-GCTGAGACATGACGAGTC-3′ (SEQ ID NO:6), at a concentration of 0.2    μM. The φ29 DNA polymerase (160 ng per 50 μl) is added and the    reaction mixture is incubated for 30 minutes at 30° C.-   3. To the above solutions a compensating buffer is added to achieve    the following concentrations of reagents: 35 mM Tris-HCl (pH 8.2), 2    mM spermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP, 333 μM    UTP, 667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03% Tween-20, 2    Units per μl of T7 RNA polymerase. The reaction is incubated for 90    minutes at 37° C.-   4. One-tenth volume of 5 M NaCl is added to the above reactions, and    the resulting solution is mixed with an equal volume of ExpressHyb    reagent (Clontech Laboratories, Palo Alto, Calif.). Hybridization is    performed by contacting the amplified RNA solution, under a cover    slip, with the surface of a glass slide (Guo et al. (1994))    containing a 2.5 mm dot with 2×10¹¹ molecules of a covalently bound    29-mer oligonucleotide with the sequence    5′-TTTTTTTTTTTCCAACCTCCATCACTAGT-3′ (SEQ ID NO:7). The last 14    nucleotides of this sequence are complementary to the amplified    mutant gene RNA, and hence the mutant RNA binds specifically.    Another 2.5 mm dot on the slide surface contains 2×10¹¹ molecules of    a covalently bound 29-mer oligonucleotide with the sequence    5′-TTTTTTTTTTTCCAACCTCGATCACTAGT-3′ (SEQ ID NO:8). The last 14    nucleotides of this sequence are complementary to the amplified wild    type gene RNA, and hence the wild type RNA binds specifically. The    glass slide is washed once with 2×SSPE as described (Guo et al.    (1994)), then washed twice with 2×SSC (0.36 M sodium saline    citrate), and then incubated with fluoresceinated avidin (5 μg/ml)    in 2×SSC for 20 minutes at 30° C. The slide is washed 3 times with    2×SSC and the slide-bound fluorescence is imaged at 530 nm using a    Molecular Dynamics Fluorimager.

Example 3 Detection of a Mutant Ornithine Transcarbamylase (OTC) GeneUsing Gap-filling LM-RCT

This example describes detection of human DNA containing a mutant form(G to C) at position 114 of exon 9 of the ornithine transcarbamylasegene (Hata et al. (1988)) using gap-filling LM-RCT. Human DNA for theassay is prepared by extraction from buffy coat using a standard phenolprocedure. In this example, two different open circle probes are used todetect the mutant and wild type forms of the gene. No gapoligonucleotide is used.

-   1. Two DNA samples (400 ng each) are heat-denatured for 4 minutes at    97° C., and incubated in the presence of one of the following    5′-phosphorylated open circle probes.

Open circle probe for mutant gene (96 nucleotides):

-   5′-TAAAAGACTTCATCATCCATCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGGGAACACTAGTGATGG-3′    (SEQ ID NO:11). When this probe hybridizes to the target sequence,    there is a gap space of seven nucleotides between the ends of the    open circle probe.

Open circle probe for wild type gene (96 nucleotides):

-   5′-TAAAAGACTTCATCATCCATCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGGGAACACTAGTGATCG-3′    (SEQ ID NO:12). When this probe hybridizes to the target sequence,    there is a gap space of seven nucleotides between the ends of the    open circle probe.

Each of the OCP-target sample mixtures are incubated in anextension-ligation mixture as described by Abravaya et al. (1995). Thereaction, in a volume of 40 μl, contains 50 mM Tris-HCl (pH 7.8), 25 mMMgCl₂, 20 mM potassium acetate, 10 μM NAD, 80 nM open circle probe, 40μM dATP, 40 μM dGTP, 1 Unit Thermus flavus DNA polymerase (lacking 3′-5′exonuclease activity; MBR, Milwaukee, Wis.), and 4000 Units Thermusthermophilus DNA ligase (Abbott laboratories). The reaction is incubatedfor 60 seconds at 85° C., and 50 seconds at 60° C. in a thermal cycler.No thermal cycling is performed. This results in hybridization of theopen circle probe to the target sequence, if present, filling in of thegap space by the T. flavus DNA polymerase, and ligation by the T.thermophilus ligase. The discriminating nucleotide in the open circleprobes above is the penultimate nucleotide. T. flavus DNA polymerase isused in the reaction to match the thermal stability of the T.thermophilus ligase.

-   2. 25 μl are taken from each of the above reactions and mixed with    an equal volume of a buffer consisting of 50 mM Tris-HCl (pH 7.5),    10 mM MgCl₂, 1 mM DTT, 400 μM each of dTTP, dATP, dGTP, dCTP; and    containing the 18-base oligonucleotide primer    5′-GCTGAGACATGACGAGTC-3′ (SEQ ID NO:6), at a concentration of 0.2    μM. The φ29 DNA polymerase (160 ng per 50 μl) is added and the    reaction mixture is incubated for 30 minutes at 30° C. to perform    rolling circle amplification catalyzed by φ29 DNA polymerase. The    Thermus flavus DNA polymerase does not significantly interfere with    rolling circle replication because it has little activity at 30° C.    If desired, the Thermus flavus DNA polymerase can be inactivated,    prior to rolling circle replication, by adding a neutralizing    antibody analogous to antibodies for blocking Taq DNA polymerase    prior to PCR (Kellogg et al., Biotechniques 16(6):1134-1137 (1994)).-   3. To each of the above solutions are added compensating buffer to    achieve the following concentrations of reagents: 35 mM Tris-HCl (pH    8.2), 2 mM spermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP,    333 μM UTP, 667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03%    Tween-20, 2 Units per μl of T7 RNA polymerase. The reactions are    incubated for 90 minutes at 37° C.-   4. One-tenth volume of 5 M NaCl is added to the each solution    containing the biotinylated RNA generated by T7 RNA polymerase, and    the resulting solution is mixed with an equal volume of ExpressHyb    reagent (Clontech laboratories, Palo Alto, Calif.). Hybridization is    performed by contacting the amplified RNA solution, under a cover    slip, with the surface of a glass slide (Guo et al. (1994))    containing a 2.5 mm dot with 2×10¹¹ molecules of a covalently bound    29-mer address probe with the sequence    5′-TTTTTTTTTTTCCAAATTCTCCTCCATCA-3′ (SEQ ID NO:13). The last 14    nucleotides of this sequence are complementary to the amplified    mutant gene RNA, and hence the mutant RNA binds specifically.    Another 2.5 mm dot on the slide surface contains 2×10¹¹ molecules of    a covalently bound 29-mer address probe with the sequence    5′-TTTTTTTTTTTCCAAATTCTCCTCGATCA-3′ (SEQ ID NO:14). The last 14    nucleotides of this sequence are complementary to the amplified wild    type gene RNA, and hence the wild type RNA binds specifically. The    glass slide is washed once with 2×SSPE as described (Guo et al.    (1994)), then washed twice with 2×SSC (0.36 M sodium saline    citrate), and then incubated with fluoresceinated avidin (5 μg/ml)    in 2×SSC for 20 minutes at 30° C. The slide is washed 3 times with    2×SSC and the slide-bound fluorescence is imaged at 530 nm using a    Molecular Dynamics Fluorimager.

Example 4 Reverse Transcription of Ornithine Transcarbamylase (OTC) mRNAFollowed by Mutant cDNA Detection Using Gap-filling LM-RCT

This example describes detection of human MRNA containing a mutant form(G to C) at position 114 of exon 9 of the ornithine transcarbamylasegene (Hata et al. (1988)) using cDNA generated by reverse transcription.RNA for the assay is prepared by TRIzol (Life Technologies, Inc.,Gaithersburg, Md.) extraction from liver biopsy.

-   1. OTC exon 9 cDNA is generated as follows:-   A liver biopsy sample is stored at −80° C. in a 0.5 ml. reaction    tube containing 40 Units of RNase inhibitor (Boehringher Mannheim).    Total RNA is extracted from the frozen sample using TRIzol reagent    (Life Technologies, Inc., Gaithersburg, Md.), and dissolved in 10 μl    water. A 19 μl reaction mixture is prepared containing 4 μl of 25 mM    MgCl₂, 2 μl of 400 mM KCl, 100 mM Tris-HCl (pH 8.3), 8 μl of a 2.5    mM mixture of dNTP's (dATP, dGTP, dTTP, dCTP), 1 μl of MuLV reverse    transcriptase (50 U, Life Technologies, Inc., Gaithersburg, Md.), 1    μl of MuLV reverse transcriptase primer    (5′-TGTCCACTTTCTGTTTTCTGCCTC-3′; SEQ ID NO:15), 2 μl of water, and 1    μl of RNase inhibitor (20 U). The reaction mixture is added to 1 μl    of the Trizol-purified RNA solution, and incubated at 42° C. for 20    minutes to generate cDNA.-   2. Two 20 μl cDNA samples from step 1 are heat-denatured for 4    minutes at 98° C., and incubated under ligation conditions in the    presence of two 5′-phosphorylated probes:

Open circle probe (95 nucleotides):

5′-ATCACTAGTGTTCCTTCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAAC (SEQ ID NO: 16)GGTCACTAATACGACTCACTATAGGGGATGATGAAGTCTTTTAT-3′

Gap probe for mutant gene (8 nucleotides):

5′-TAGTGATG-3′

Gap probe for wild type gene (8 nucleotides):

5′-TAGTGATC-3′T4 DNA ligase (New England Biolabs) is added at a concentration of 5units per μl, in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.20 MNaCl, 10 mM MgCl₂, 2 mM ATP. The concentration of open circle probe is80 nM, and the concentration of gap oligonucleotide is 100 nM. The totalvolume is 40 μliters. Ligation is carried out for 25 minutes at 37° C.

-   3. 25 μl are taken from each of the above reactions and mixed with    an equal volume of a buffer consisting of 50 mM Tris-HCl (pH 7.5),    10 mM MgCl₂, 1 mM DTT, 200 μM each of dTTP, dATP, dGTP, dCTP; and    containing the 18-base rolling circle replication primer    5′-GCTGAGACATGACGAGTC-3′ (SEQ ID NO:6), at a concentration of 0.2    μM. The φ29 DNA polymerase (160 ng per 50 μl) is added and the    reaction mixtures are incubated for 30 minutes at 30° C.-   4. To the above solutions are added compensating buffer to achieve    the following concentrations of reagents: 35 mM Tris-HCl (pH 8.2), 2    mM spermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP, 333 μM    UTP, 667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03% Tween-20, 2    Units per μl of T7 RNA polymerase. The reaction is incubated for 90    minutes at 37° C.-   5. One-tenth volume of 5 M NaCl is added to the each solution    containing the biotinylated RNA generated by T7 RNA polymerase, and    the resulting solution is mixed with an equal volume of ExpressHyb    reagent (Clontech laboratories, Palo Alto, Calif.). Hybridization is    performed by contacting the amplified RNA solution, under a cover    slip, with the surface of a glass slide (Guo et al. (1994))    containing a 2.5 mm dot with 2×10¹¹ molecules of a covalently bound    29-mer address probe with the sequence    5′-TTTTTTTTTTTTTTTTGATGGAGGAGAAT-3′ (SEQ ID NO:17). The last 14    nucleotides of this sequence are complementary to the amplified    mutant gene RNA, and hence the mutant RNA binds specifically.    Another 2.5 mm dot on the slide surface contains 2×10¹¹ molecules of    a covalently bound 29-mer address probe with the sequence    5′-TTTTTTTTTTTTTTTTGATCGAGGAGAAT-3′ (SEQ ID NO:9). The last 14    nucleotides of this sequence are complementary to the amplified wild    type gene RNA, and hence the wild type RNA binds specifically. The    glass slide is washed once with 2×SSPE as described (Guo et al.    (1994)), then washed twice with 2×SSC (0.36 M sodium saline    citrate), and then incubated with fluoresceinated avidin (5 μg/ml)    in 2×SSC for 20 minutes at 30° C. The slide is washed 3 times with    2×SSC and the slide-bound fluorescence is imaged at 530 nm using a    Molecular Dynamics Fluorimager.

Example 5 Multiplex Immunoassay Coupled to Rolling Circle Amplification

This example describes an example of multiplex detection of differenttarget molecules using reporter antibodies. The signal that is detectedis produced by rolling circle amplification of the target sequenceportion of the reporter antibodies.

-   1. Three different monoclonal antibodies, each specific for a    different target molecule, are coupled to three different arbitrary    DNA sequences (A, B, C) that serve as unique identification tags    (target sequences). In this example, the three antibodies are    maleimide-modified and are specific for β-galactosidase, hTSH, and    human chorionic gonadotropin (hCG). The antibodies are coupled to    aminated DNA oligonucleotides, each oligonucleotide being 50    nucleotides long, using SATA chemistry as described by Hendrickson    et al. (1995). The resulting reporter antibodies are called reporter    antibody A, B, and C, respectively.-   2. Antibodies specific for the target molecules (not the reporter    antibodies) are immobilized on microtiter dishes as follows: A 50 μl    mixture containing 6 μg/ml of each of the three antibodies in sodium    bicarbonate (pH 9) is applied to the wells of a microtiter dish,    incubated overnight, and washed with PBS-BLA (10 mM sodium phosphate    (pH 7.4), 150 mM sodium chloride, 2% BSA, 10% β-lactose, 0.02%    sodium azide) to block non-adsorbed sites.-   3. Serial dilutions of solutions containing one or a combination of    the three target molecules (hTSH, hCG, and β-galactosidase) are    added to the wells. Some wells are exposed to one target molecule, a    mixture of two target molecules, or a mixture of all three target    molecules. After 1 hour of incubation, the wells are washed three    times with TBS/Tween wash buffer as described by Hendrickson et al.    (1995).-   4. Fifty microliters of an appropriately diluted mixture of the    three reporter antibodies (A+B+C) are added to each well of the    microtiter dish. The plate is incubated at 37° C. for 1 hour, and    then washed four times with TBS/Tween buffer.-   5. To each well is added a mixture of three pairs of open circle    probes and gap oligonucleotides, each pair specific for one of the    three target sequence portions of the reporter antibodies. In this    example, the open circle probes have the same spacer region of 49    bases including a universal primer complement portion, and different    18 nucleotide target probe portions at each end. Each cognate pair    of open circle probe and gap oligonucleotide is designed to    hybridize to a specific target sequence (A, B, or C) in the target    sequence portion of the reporter antibodies. Specifically, Open    circle probe A′ has left and right target probe portions    complementary to two 18-base sequences in tag sequence A separated    by 8 bases that are complementary to the 8-nucleotide gap    oligonucleotide A′. The same is the case for open circle probe and    gap oligonucleotide pairs B′ and C′. The concentration of each open    circle probe is 80 nM, and the concentration of each gap    oligonucleotide is 120 nM.-   6. T4 DNA ligase (New England Biolabs) is added to each microtiter    well at a concentration of 5 units per μl, in a reaction buffer    consisting (10 mM Tris-HCl (pH 7.5), 40 mM potassium acetate, 10 mM    MgCl₂, 2 mM ATP). The total volume in each well is 40 μliters.    Ligation is carried out for 45 minutes at 37° C.-   7. To each microtiter well is added 20 μl of a compensating solution    containing dTTP, dATP, dGTP, dCTP (400 μM each), the universal    18-base oligonucleotide primer 5′-GCTGAGACATGACGAGTC-3′ (SEQ ID    NO:6) (at a final concentration of 0.2 μM), and φ29 DNA polymerase    (at 160 ng per 50 μl). The reaction for 30 minutes at 30° C.-   8. After incubation, a compensating buffer is added to each well to    achieve the following concentrations of reagents: 35 mM Tris-HCl (pH    8.2), 2 mM spermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP,    333 μM UTP, 667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03%    Tween-20, 2 Units per μl of T7 RNA polymerase. The reaction is    incubated for 90 minutes at 37° C., generating biotinylated RNA.-   9. One-tenth volume of 5 M NaCl is added to each well, and the    resulting solution is mixed with and equal volume of ExpressHyb    reagent (Clontech laboratories, Palo Alto, Calif.). Hybridization is    performed by contacting the mixture of amplified RNAs, under a cover    slip, with the surface of a glass slide containing three separate    dots of 2×10¹¹ molecules of three different covalently bound 31-mer    oligonucleotides (A, B, C) (Guo et al. (1994)). The last 16 bases of    each oligonucleotide are complementary to a specific segment (4    bases+8 bases+4 bases), centered on the 8-base gap sequence, of each    of the possible amplified RNAs generated from tag sequences A, B,    or C. Hybridization is carried out for 90 minutes at 37° C. The    glass slide is washed once with 2×SSPE as described (Guo et al.    (1994)), then washed twice with 2×SSC (0.36 M sodium saline    citrate), and then incubated with fluoresceinated avidin (5 μg/ml)    in 2×SSC for 20 minutes at 30° C. The slide is washed 3 times with    2×SSC and the surface-bound fluorescence is imaged at 530 nm using a    Molecular Dynamics Fluorimager to determine if any of tag sequences    A or B or C was amplified.

All publications cited herein are hereby incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method comprising a DNA ligation operation and an amplificationoperation, wherein the DNA ligation operation comprises circularizationof an open circle probe, wherein circularization of the open circleprobe is dependent on hybridization of the open circle probe to a targetsequence, wherein the amplification operation comprises a) rollingcircle replication of the circularized open circle probe to producetandem sequence DNA and b) secondary DNA strand displacement, whereinsecondary DNA strand displacement is accomplished by hybridizingsecondary DNA strand displacement primers to the tandem sequence DNAproduced by the rolling circle replication of the circularized opencircle probe to produce secondary tandem sequence DNA, and wherein thetandem sequence DNA as well as the secondary tandem sequence DNA isdetected.
 2. The method of claim 1 wherein the tandem sequence DNA isdetected using molecular beacon detection probes.
 3. The method of claim1 wherein the tandem sequence DNA is detected using detection probeslabeled with fluorescent moieties where the fluorescent moietiesfluoresce only when the detection probe is hybridized, wherein thedetection probes hybridize to the tandem sequence DNA.
 4. The method ofclaim 1 wherein the target sequence is coupled to a specific bindingmolecule, wherein the specific binding molecule can interact with atarget molecule.
 5. The method of claim 4 wherein the target molecule isa protein.
 6. The method of claim 4 wherein the target molecule is anucleic acid molecule.
 7. A method comprising a) tethering anoligonucleotide portion of a reporter binding agent to a specificbinding molecule portion of the reporter binding agent, wherein theoligonucleotide portion of the reporter binding agent is anamplification target circle; and b) performing an amplificationoperation, wherein the specific binding molecule portion of the reporterbinding agent binds to a target molecule, wherein the oligonucleotideportion of the reporter binding agent is not the target molecule, andwherein the amplification operation comprises amplification of theamplification target circle via rolling circle replication to producetandem sequence DNA.
 8. The method of claim 7 wherein the tandemsequence DNA is detected using molecular beacon detection probes.
 9. Themethod of claim 7 wherein the tandem sequence DNA is detected usingdetection probes labeled with fluorescent moieties where the fluorescentmoieties fluoresce only when the detection probe is hybridized, whereinthe detection probes hybridize to the tandem sequence DNA.
 10. Themethod of claim 7 wherein the target molecule is a protein.
 11. Themethod of claim 7 wherein the target molecule is a nucleic acidmolecule.
 12. The method of claim 7 wherein the specific bindingmolecule is an antibody.
 13. A method comprising a DNA ligationoperation and an amplification operation, wherein the DNA ligationoperation comprises circularization of an open circle probe, whereincircularization of the open circle probe is dependent on hybridizationof the open circle probe to a target sequence, wherein the amplificationoperation comprises rolling circle amplification of the circularizedopen circle probe to produce tandem sequence DNA, wherein the tandemsequence DNA is detected, wherein rolling circle amplification is primedby a rolling circle replication primer, a secondary DNA stranddisplacement primer, and a tertiary DNA strand displacement primer,wherein the rolling circle replication primer and tertiary DNA stranddisplacement primer are complementary to the circularized open circleprobe, wherein the secondary DNA strand displacement primer iscomplementary to sequences in the tandem sequence DNA, wherein thesecondary DNA strand displacement primer is not complementary to thecomplement of either the rolling circle replication primer or thetertiary DNA strand displacement primer.
 14. The method of claim 13wherein the tandem sequence DNA is detected using molecular beacondetection probes.
 15. The method of claim 13 wherein the tandem sequenceDNA is detected using detection probes labeled with fluorescent moietieswhere the fluorescent moieties fluoresce only when the detection probeis hybridized, wherein the detection probes hybridize to the tandemsequence DNA.
 16. The method of claim 13 wherein the target sequence iscoupled to a specific binding molecule, wherein the specific bindingmolecule can interact with a target molecule.
 17. The method of claim 16wherein the target molecule is a protein.
 18. The method of claim 16wherein the target molecule is a nucleic acid molecule.
 19. A methodcomprising an amplification operation, wherein a reporter binding agentand a target molecule are brought into contact, wherein the reporterbinding agent comprises a specific binding molecule and anoligonucleotide, wherein the specific binding molecule interacts withthe target molecule, wherein an amplification target circle and thereporter binding agent are brought into contact, wherein theamplification operation comprises rolling circle amplification of theamplification target circle to produce tandem sequence DNA, wherein thetandem sequence DNA is detected, wherein rolling circle amplification isprimed by the oligonucleotide of the reporter binding agent, a secondaryDNA strand displacement primer, and a tertiary DNA strand displacementprimer.
 20. The method of claim 19 wherein the tandem sequence DNA isdetected using molecular beacon detection probes.
 21. The method ofclaim 19 wherein the tandem sequence DNA is detected using detectionprobes labeled with fluorescent moieties where the fluorescent moietiesfluoresce only when the detection probe is hybridized, wherein thedetection probes hybridize to the tandem sequence DNA.
 22. The method ofclaim 19 wherein the target molecule is a protein.
 23. The method ofclaim 19 wherein the target molecule is a nucleic acid molecule.
 24. Amethod comprising an amplification operation, wherein a reporter bindingagent and a target molecule are brought into contact, wherein thereporter binding agent comprises a specific binding molecule and anoligonucleotide, wherein the oligonucleotide comprises an amplificationtarget circle, wherein the specific binding molecule interacts with thetarget molecule, wherein the amplification operation comprises rollingcircle amplification of the amplification target circle to producetandem sequence DNA, wherein the tandem sequence DNA is detected,wherein rolling circle amplification is primed by a rolling circlereplication primer, a secondary DNA strand displacement primer, and atertiary DNA strand displacement primer, wherein the rolling circlereplication primer and tertiary DNA strand displacement primer, arecomplementary to the amplification target circle, wherein the rollingcircle replication primer and tertiary DNA strand displacement primerare not complementary to the same sequence on the amplification targetcircle, wherein the secondary DNA strand displacement primer iscomplementary to sequences in the tandem sequence DNA, wherein thesecondary DNA strand displacement primer is not complementary to thecomplement of either the rolling circle replication primer or thetertiary DNA strand displacement primer.
 25. The method of claim 24wherein the tandem sequence DNA is detected using molecular beacondetection probes.
 26. The method of claim 24 wherein the tandem sequenceDNA is detected using detection probes labeled with fluorescent moietieswhere the fluorescent moieties fluoresce only when the detection probeis hybridized, wherein the detection probes hybridize to the tandemsequence DNA.
 27. The method of claim 24 wherein the target molecule isa protein.
 28. The method of claim 24 wherein the target molecule is anucleic acid molecule.